4 Dear Colleagues and Friends, We are honored to invite you to Molecular Immunology & Immunogenetics Congress (MIMIC2012) that will be held in Antalya, Turkey, from April 27 to 29, 2012 under the auspices of the Turkish Society of Immunology. The venue of the congress will be the Papillon Aysha Hotel in Antalya, a beautiful historical city and holiday resort on the shores of the Mediterranean Sea. This congress will cover modern aspects of molecular and applied immunology as well as immunogenetics. Since the main aim of MIMIC2012 is to connect international scientists and immunologists to encourage new collaborations in the field of molecular immunology and immunogenetics, we invite scientists from all over the world to present their studies and to discuss the new findings and discoveries with other researchers. Additionally, we are pleased to announce that the organizing committee has made special efforts to offer a large number of grants and awards covering registration, accommodation and part of travel expenses. Therefore, young researchers are particularly encouraged to apply for attending to the congress. Nationally and internationally renowned speakers will be giving talks during the congress. Simultaneous translation to Turkish will be available for Turkish participants. We are looking forward to seeing you in Antalya for this unique and stimulating event! On behalf of the Organizing Committee, Prof. H. Barbaros ORAL Congress President Prof. Gunnur DENIZ President of TSI 4

30 Innate immunity and malaria parasites Cevayir Coban Laboratory of Malaria Immunology, Immunology Frontier Research Center, Osaka University Malaria is responsible for the deaths of over a million people each year. Approximately half the world's population is at potential risk of the disease, which infects 300 million people annually. The Plasmodium parasites responsible for the disease have a complex life cycle which involves both a vertebrate (mammal) and invertebrate (Anopheles mosquito) host. The parasites have evolved many strategies that allow them to evade the host immune system. The innate immune system is a highly specialized first line defense system against many infectious diseases, including malaria. However, the role of innate immunity in response to Plasmodium parasites and protective mechanism (s) elucidated by host have not been identified thoroughly. We ve recently identified the unknown role of Lipocalin 2, a TLR-mediated antibacterial protein, in malaria infection. We ve found that Lipocalin 2 helps to control parasitemia levels during blood-stage malaria infection via controlling iron homeostasis. Manipulation of iron metabolism of host is one of the important weapons for invading pathogens, because iron is an essential element for the growth of all living organisms. I ll discuss about this new finding and its implications in my talk. Recommended readings 1. Coban C, Ishii KJ, Horii T, Akira S. Manipulation of host innate immune responses by the malaria parasite. Trends Microbiol. 2007; 15: Doolan DL, Dobano C, Baird JK. Acquired immunity to malaria. Clin Microbiol Rev. 2009; 22(1): Nairz M, Schroll A, Sonnweber T, Weiss G. The struggle for iron - a metal at the host-pathogen interface. Cell Microbiol. 2010; 12: Wang J, Pantopoulos K. Regulation of cellular iron metabolism. Biochem J. 2011; 434: Flo TH, Smith KD, Sato S, Rodriguez DJ, Holmes MA, Strong RK, Akira S, Aderem A. Lipocalin 2 mediates an innate immune response to bacterial infection by sequestrating iron. Nature. 2004; 432:

34 How does the innate immune system deal with the microbial metagenome? Şefik Ş. Alkan, Ph.D. Alkan Consulting, Basel, Switzerland and UMDNJ, Medical School, Dept. Molecular Genetics & Microbiology, Piscataway, USA One would think that to be a human being, on a biological level, should be determined by our own genome. But living in and on our body is an ecosystem of microorganisms that outnumber our own cells by at least a factor of ten. Neonates emerge from a sterile uterine environment are instantly colonized by microbes from the immediate environment. These microbes reside on our skin (1 trillion), in our nose/mouth (100 million), and in our gut (100 trillion). That means we carry 100 times as many microbial genes (microbiome), as human genes with our bodies. Does that mean that microbial DNA may contain even more information about us than does our own DNA? It is likely that, early childhood exposure or the lack of exposure to certain microbiome (i.e. lack of information exchange) have important implications for health and disease later in life. Recent technological advances, mainly in the field of metagenomics, (study of genetic material recovered directly from environmental samples, such as 16S rrna) are rapidly enriching our knowledge of the genomes and functions of many of these microbial communities. The ultimate aim is to discover how perturbations of the microbiome might be related to various diseases, including inflammatory bowel disease, asthma, and obesity. A recent study for examples illuminates the role of intestinal bacteria in the regulation of lipid metabolism via IgA. Other research focuses on the potential role of microbes in anxiety, depression, and autism. These findings will have the potential to change the landscape of medicine in the near future. In the meantime, we need to solve a scientific puzzle; how does the immune system with only 1 trillion cells, manage to create such diverse responses to cope with the ever changing and evolving microbial world with 100 times more genome? I will try to answer this major question throughout this presentation. I will first summarize the cells and molecules of innate immunity, which recognizes the presence of invaders instantly, by evolutionally conserved sensing receptors such as TLRs, NLRs, RLRs and CLRs. The quick innate response to the signature structure of microbes alarms and shapes the second wave of immunological responses. Although slow in action, the adaptive immunity has powerful ways of generating diversity, specificity and memory. As a whole the immune system needs to be collaborative, adaptable, tolerant to self and highly regulatory. Understanding our interaction with metagenome will require a holistic approach such as systems biology. References 1. Burcelina, R, et al. Immuno-microbiota cross and talk: The new paradigm of metabolic diseases. Seminars in Immunology. 2012; 24: Duerr CU, Hornef MW. The mammalian intestinal epithelium as integral player in the establishment and maintenance of host microbial homeostasis. Seminars in Immunology. 2012; 24: Natalia Shulzhenko et al. Crosstalk between B lymphocytes, microbiota and the intestinal epithelium governs immunity versus metabolism in the gut. Nature 34

35 medicine, 2011; 17: O Doherty, K. Who Are We Really? Manipulating the human microbiome has ethical implications. The Scientist, March (2012) 5. Vaarala O, Is the origin of type 1 diabetes in the gut? Immunology and Cell Biology. 2012; 90:

36 René van Lier René van Lier studied medicine at the University of Amsterdam ( ), and obtained his PhD in 1988 at the same university on research aimed to characterize properties of cell surface receptors expressed on human T cells. He continued working in the field of immunology at CLB first as a post-doc later as a group leader (now Sanquin). In 2000, he was appointed as Professor of Experimental Immunology and Head of the Department of Experimental Immunology at Academic Medical Center (AMC) in Amsterdam. In 2010 he became director of research and member of the executive board at Sanquin Blood Supply Foundation. The focus of his research is on cellular immunology and encompasses work in both human systems and in genetically modified mice. He is the chairman of the Dutch Society for Immunology, is a council member of International Union of Immunological Societies and is a member of the board of directors of EEIG ECI-EFIS. He serves at the scientific advisory boards of the Dutch Asthma foundation, the MS research foundation and the Landsteiner foundation for blood transfusion research.he has published more than 250 peer-reviewed articles. Recent Publications 1. van Gisbergen KP, Klarenbeek PL, Kragten NA., Unger PP, Nieuwenhuis MB, Wensveen FM, Ten BA, Tak PP, Eldering E, Nolte MA et al. The costimulatory molecule CD27 maintains clonally diverse CD8(+) T cell responses of low antigen affinity to protect against viral variants. Immunity. 2011; 35: Piet B, de Bree GJ, Smids-Dierdorp BS, van der Loos CM, Remmerswaal EB, von der Thusen JH, van Haarst JM, Eerenberg JP, Ten BA, van der Bij W et al. CD8 T cells with an intraepithelial phenotype upregulate cytotoxic function upon influenza infection in human lung. J Clin Invest. 2011; 121: Hertoghs KM, Moerland PD, van SA, Remmerswaal EB, Yong SL, van de Berg PJ, van Ham SM, Baas F, Ten Berge IJ and van Lier RA. Molecular profiling of cytomegalovirus-induced human CD8+ T cell differentiation. J Clin Invest. 2010; 120:

37 Identification of a BLIMP-1 homologue that regulates effector functions in T, NK and NKT cells Rene A.W. van Lier 1,2, Natasja A.M. Kragten 1,2, Kirsten M.L. Hertoghs 2, Jörg Hamann 2, Martijn A. Nolte 1, and Klaas P.J.M. van Gisbergen 1,2 1 Department of Hematopoiesis, Sanquin Research at CLB and Landsteiner Laboratory, 2 Department of Experimental Immunology, Academic Medical Center Amsterdam, The Netherlands. Latent infection with human cytomegalovirus (HCMV) induces a strong increase in the number of circulating resting, effector-type CD8+ T cells with constitutive cytolytic activity. By microarray analysis we searched for transcription factors that regulate the generation and homeostasis of these cells. Next to a number of transcription factors known to be involved in murine T H 1 and cytotoxic T cell differentiation (e.g. T-bet, Eomes and Blimp-1) we found a novel zinc-finger protein that is highly upregulated in resting cytolytic CD8 + T cells (Hertoghs et al., J Clin Invest, 2010; 120: ). Because of its close homology to BLIMP-1, a broadly expressed transcription factor involved in the regulation of B and T cell effector functions, the molecule was named HOBIT (for Homologue of BLIMP-1 in T cells). In contrast to BLIMP-1, the expression of HOBIT is restricted to immune cells endowed with cytolytic function (e.g. cytolytic CD4 + and CD8 + cells and NK cells). HOBIT is induced in CD8 + virus-specific T cells already early in the primary CMV response and in vitro studies implicated a role for IL27 in this upregulation. Three HOBIT splice variants were identified in cytolytic cells. Overexpression of a HOBIT isoform that has an identical zinc finger alignment as BLIMP-1 strongly enhanced IFN production in NK cell lines. In contrast, knock- Interestingly, in mice Hobit was found to be specifically expressed at high levels in NKT cells but hardly in conventional T cells or NK cells. Using Hobit-deficient mice, we found that Hobit was essential for the accumulation of thymic mature NKT cells. Moreover, Hobit was downregulated after antigenic stimulation in peripheral NKT cells and acted as a repressor of expansion. In contrast, after innate stimulation Hobit was required for upregulation of granzyme B in NKT cells. Thus, the novel transcription factor Hobit is critical for murine NKT cells to mediate cytotoxic responses depending on antigenic and inflammatory stimuli. In summary, although the expression markedly differs between humans and mice, in both species the newly identified transcription factor Hobit appears to have a conserved role in the regulation of lymphocyte effector functions. 37

40 New B regulatory cell subsets Willem van de Veen, MSc,a Barbara Stanic PhD,a Görkem Yaman, MD,b Marcin Wawrzyniak,a Stefan Söllner, Sci Tec,a Beate Rückert, Sci Tec,a Deniz Akdis,a Cezmi A. Akdis, MD,a and Mübeccel Akdis, MD, PhDa a Swiss Institute of Allergy and Asthma Research (SIAF), University of Zürich, Davos, CH-7270,Switzerland b Department of Medical Microbiology, Acıbadem University, Istanbul, Turkey B cells contribute to immune responses essentially through antigen presentation to T cells, secretion of cytokines and production of antibodies after differentiation to plasma cells. When they receive the right survival signals plasma cells can reside for many years in dedicated niches in the bone marrow and continuously produce antibodies independent of exposure to antigen. Upon activation IgM+IgD+ naïve B cells may undergo class switch recombination (CSR) leading to the expression of IgA, IgG or IgE antibodies. Human B cells express several toll like receptors (TLR) including TLR1, 6, 7, 8, 9 and 10. TLR7 (activated by single stranded RNA) and TLR9 (activated by hypomethylated CpG DNA) are the highest expressed TLRs on B cells. IL-10 is a key regulator of inflammatory responses and protects the host from tissue damage as a result of excessive inflammation. It suppresses antigen presentation through downregulation of class II major histocompatibility complex molecules and co-stimulatory molecules on antigen presenting cells. Furthermore it suppresses the production of pro-inflammatory chemokines and cytokines. On the other hand, IL-10 enhances the survival, proliferation, differentiation and isotype switching of human B cells. IL-10 augments IgG4 production, whereas it inhibits IL-4- induced IgE CSR. IL-10-mediated immunosuppressive functions of B cells have been described in murine models of autoimmunity, infection, and cancer. The relevance of immune regulatory functions of human B cells is illustrated by patients treated for rheumatoid arthritis with the B cell depleting antibody rituximab who showed exacerbation of ulcerative colitis and development of psoriasis. Interestingly, an increase in IL-10-producing B cells also occurs during ultra rush high dose allergenspecific immunotherapy of venom allergic individuals by bee venom (BV-SIT). Regulatory B cells expressing IL-10 suppress immune responses and the lack or loss of regulatory B cells leads to exacerbated symptoms in experimental autoimmune encephalitis, chronic colitis, contact hypersensitivity, collagen-induced arthritis and non-obese diabetic mouse models. Another B cell-related immune regulatory response restricted to humans is induction of non-inflammatory IgG4 antibodies, which is characteristic for high dose antigen tolerance models. Several molecules including CD25 and PD-L1 were upregulated in IL-10-producing B cells. Br1 cells potently suppressed antigen-specific CD4+ T cell proliferation whereas other B cells did not. Furthermore we demonstrate that human Br1 cells show selectively increased production of IgG4. B cells specific for the major bee venom allergen phospholipase A2 that were isolated from beekeepers had increased expression of IL-10 and IgG4. Human Br1 cells may regulate humoral and cellular immunological tolerance through suppression of T cells responses and production of anti-inflammatory IgG4 antibodies. 40

41 Moncef Zouali M. Zouali completed his post-graduate studies in Immunology at the University of Paris and the Institut Pasteur in Paris (France). As a research associate at Tufts University Medical School (Boston, MA. USA), his interest in autoimmunity was cultivated. Currently, he is a "Director of Research" at the Inserm, the French National Medical Council in Paris, and the University of Paris Diderot, Sorbone Paris Cité. In addition to publishing articles in professional journals, M. Zouali has edited several books. The last recently published volume is entitled Epigenetics of Autoimmune diseases". M. Zouali serves on the board of scientific journals, and acts as reviewer for international granting bodies. M. Zouali received national and international awards for his work on autoimmune diseases. Among his professional honors, he received a John F. Fogarty International Award (National Institutes of Health, Washington, DC, USA) and a Senior Scholar Fulbright Award (Washington, DC, USA). He also was the recipient of the French Medical Foundation Award on Autoimmune Diseases, and the Khwarizmi International Award in Medical Sciences (UNESCO, WIPO, and IROST). Selected Publications 1. Zouali M. Taming Lupus. Scientific American. 2005; 292: Viau M, Longo NS, Lipsky PE, and Zouali M. Staphylococcal protein a deletes B-1a and marginal zone B lymphocytes expressing human immunoglobulins: an immune evasion mechanism. J Immunol. 2005; 175, Mazari L, Ouarzane M, and Zouali M. Subversion of B lymphocyte tolerance by hydralazine, a potential mechanism for drug-induced lupus. Proc Natl Acad Sci USA. 2007; 104, Zouali M. Receptor editing and receptor revision in rheumatic autoimmune diseases. Trends Immunol. 2008; 29: Hikada M, and Zouali M. Multistoried roles for B lymphocytes in autoimmunity. Nature Immunology. 2010; 11: Yim S, Chung Y, Jin E, Shim S, Kim J, Kim Y, Shin S, Pae H, Chung H, and Zouali M. Copy number variation of the VPREB1 gene in rheumatoid arthritis affects negative selection of self-reactive B cells. Mol Immunol. 2011; 48:

43 Moshe Arditi Executive Vice Chair, Research, Department of Pediatrics Director, Division of Pediatric Infectious Diseases, Allergy and Immunology Moshe Arditi, MD, is Executive Vice-Chair of Research in the Department of Pediatrics, Director of the Division of Pediatric Infectious Diseases, Allergy and Immunology at Cedars-Sinai Medical Center. Prior to joining Cedars- Sinai, Dr. Arditi was an Associate Professor of Pediatrics at Childrens Hospital Los Angeles. Dr. Arditi's major area of research interest is the molecular pathogenesis of inflammation, sepsis and septic shock as it relates to overwhelming bacterial infection. The National Institutes of Health (NIH) and the American Heart Association support his research projects on toll-like receptors. Dr. Arditi is a member of the Society of Pediatric Research, American Academy of Pediatrics, American Society of Microbiology, Infectious Disease Society and the International Endotoxin Society. In addition, he serves as a reviewer to the NIH Study Section Bacteriology Mycology. Dr. Arditi received his medical degree from Istanbul University, School of Medicine. He completed his internship and residency in pediatrics at the University of Chicago School of Medicine and a pediatric fellowship at Northwestern University Children's Memorial Hospital, where he also served as a research associate. 43

44 ABO Incompatible Renal Transplantation Prof. Dr. Aydın Turkmen Istanbul School of Medicine Nephrology Department Organ shortage is a big problem in all over the world. Because of this serious problem, ABO-incompatible renal transplantation has been seen the alternative solving methods. ABO incompatible kidney transplantation was considered contraindication in the past. In recent years, successfull ABO-incompatible kidney transplantation may be possible by the developmentof desensitisation therapies. The first report on ABO incompatible kidney transplantation was published in 1955 by Chung et al. This extraordinary methods of kidney transplantation carry a lot of risks. It is not a simple method. It is highly expensive and having a serious clinical risks. Major problem is the significant early risk of antibody mediated rejection. The development of the desensitisation practice and the immunology has allowed increasingly successful ABO incompatible kidney transplantation. The current approach to desensitisation of blood group antigens needs the combined treatment strategies. Plasmapheresis, rituximab, IVIG and classical immunsupressive medication consisting of FK, MMF and steroids mostly used therapeutic agents. Plasmapheresis is the major method for the antibody depletion. Plasma exchange, double filtration plasmapheresis and antigen spesific immunoadsorbtion are different ways for the antibody removal. In ABO-incompatible kidney transplantation; current immunosuppressive protocol allow the similar graft survival with living kidney transplantation. The long term results of ABO incompatible kidney transplantation is equivalent to ABO compatible living kidney transplantation by the Sweden, USA and Japanese centers. As a result, ABO incompatibility should not be considered absolute contrindication of kidney transplantation. In spite of the encouraging long term results, it should not be forgotton that it is an expensive and complex approach. Then ABO incompatible kidney transplantation should be performed by experienced transplant centers. 44

45 Hiroshi Kiyono Dean and Professor Division of Mucosal Immunology Department of Microbiology and Immunology The Institute of Medical Science The University of Tokyo Shirokane-dai, Minato-ku, Tokyo JAPAN Tel: Fax: Dr. Hiroshi Kiyono, D.D.S., Ph. D is a Dean, the Institute of Medical Science, the University of Tokyo and Professor, Department of Microbiology and Immunology, at the same institute. At same time, he is also holding an Adjunct Professorship at the University of Alabama at Birmingham (UAB) and a Clinical Professorship at Arizona State University in USA. He has had a distinguished career in studying the molecular and cellular mechanisms controlling mucosal immune responses to infection and inflammation in the both digestive and respiratory tracts. Dr. Kiyono obtained his dental degree (DDS) from Nihon University, Japan and Ph.D from UAB. His background as a dentist combined with extensive research experience in the field of Mucosal Immunology at UAB, Max-Plank Institute, Osaka University and now, the University of Tokyo make him exceptionally well qualified to discuss the current and future direction of mucosal immunology for the development of mucosal vaccine and mucosal immunotherapy for the control of infectious and immunological diseases. To reflect his scientific contribution, he has been listed in ISI Highly Cited Researchers List since He is the past President of Society for Mucosal Immunology. For the recognition of his scientific contribution in the area of Mucosal Immunology and Mucosal Vaccine development, he received of several prestigious awards including NIH New Investigator Research Award, NIH Research Career Development Award, The Japanese Society for Vaccinology Takahashi Award, and Hideyo Noguchi Memorial Medical Science Award. He has a total of 400 publications in peer review journals and edited a total of 20 books. Selected Publications 1. Kunisawa J, Kurashima Y, & Kiyono H. Gut-associated lymphoid tissues for the development of oral vaccines. Adv Drug Deliv Rev., in press. 2. Goto Y, & Kiyono H. Epithelial barrier: an interface for the cross-communication between gut flora & immune system. Immunol Rev. 2012; 245, Nochi T, Yuki Y, Takahashi H, Sawada SI, Mejima M, Kohda T, Harada N, Kong IG, Sato A, Kataoka N, Tokuhara D, Kurokawa S, Takahashi Y, Tsukada H, Kozaki S, Akiyoshi K, & Kiyono H. Nanogel antigenic protein-delivery system for adjuvant-free intranasal vaccines. Nat Mater. 2010; 9: Tokuhara D, Yuki Y, Nochi T, Kodama T, Mejima M, Kurokawa S, Takahashi Y, Nanno, M, Nakanishi U, Takaiwa F, Honda T, & Kiyono H. Secretory IgAmediated protection against V. cholerae and heat-labile enterotoxin-producing enterotoxigenic Escherichia coli by rice-based vaccine. Proc Natl Acad Sci USA. 45

49 The efficacy of insulin gene therapy in autoimmune diabetes Salih Sanlioglu VMD, PhD Human Gene and Cell Therapy Center of Akdeniz University Hospitals and Clinics, Akdeniz University Faculty of Medicine, Antalya, Turkey, Diabetes mellitus is characterized by the elevated blood glucose levels as a result of insulin deficiency or failure in the uptake of glucose into peripheral tissues due to insulin resistance. Today, diabetes is the third most common disease and the fourth leading cause of death in North America (1). Since prolonged exposure to hyperglycemia can cause vascular complications leading to multiple organ failure in the long run, controlling glycemia is vital to avert diabetes and put off chronic complications. Therefore, insulin is indicated for patients with T1D or with T2D, especially when the combination of exercise, diet and oral antiglycemic agents is not adequate in maintaining glycemic control. However, physiologic insulin secretion cannot be compensated just by insulin administration into the patients. Although, the best cure for T1D patients is the pancreas transplantation, scarcity of donors, the need for major surgery and life long immunosuppression make it less practical for application (2). Pancreatic islet cell transplantation offers an alternative means to restore insulin expression for prolonging and improving the quality of life in T1D patients (3). However, shortage of organ donors greatly limits its use in clinics (4). In addition, current islet transplantation protocols including Edmonton protocol require life long immune suppression. Unfortunately, despite the initial success obtained in islet transplantation, high frequency of non-functioning grafts and secondary graft failure require patients to resume insulin administration at 5 years (5). Consequently, novel gene and cell therapy approaches for diabetes are needed as an alternative avenue to overcome these barriers in diabetic patients (6). Because insulin deficiency is the main sequel of diabetes, transfer of insulin gene to restore insulin deficiency by gene therapy, appears to be an attractive treatment modality even though the disease itself is not caused by a single genetic defect. There are two gene delivery methods available, one of which is the ex vivo approach involving the removal of cells from the patients, modifying it outside the body and transplanting it back to the patients. In vivo method, however, concerns direct injection of gene transfer vectors to the patients through various routes (intravenous, subcutaneous, etc). Because of its simplicity and convenience, in vivo gene therapy is a favored method of gene delivery. In vivo gene therapy approaches involved in lowering of blood glucose are mainly directed at facilitating glucose utilization and concurrently inhibiting hepatic glucose production. Alternatively, transfer of genes encoding transcription factors with the potential to generate beta cells in organs like liver has also been tried. For example, transdifferentiation of hepatocytes into insulin secreting cells using gene transfer of the transcription factors such as Pancreatic/Duodenal Homeobox Gene 1 (PDX1) and NeuroD into hepatocytes ameliorated STZ induced diabetes in mice (7). Furthermore, NeuroD and Betacellulin mediated gene therapy approach induced islet neogenesis, generating insulin-producing cells in liver (8). Clearly, insulin gene delivery through gene transfer vectors represents one potential avenue to mimic endogenous insulin expression profile in diabetic patients. Thus, the main goals of insulin gene therapy can be defined as to restore insulin 49

55 Chiara Bonini Dr. Chiara Bonini graduated in Medicine at the University of Milan, Italy in In 1998 she received the European Board in Hematology at the University of Pavia. In 1993 she worked as an intern in the Bone Marrow Transplant Unit at the Memorial Sloan Kettering Cancer Center, New York. Between 1994 and 1998 she worked as a Medical Fellow and Research Fellow at the Bone Marrow Transplant Unit and Gene Therapy Program of HSR. From 1998 to 2000 she was Senior Fellow in the Progam in Immunology at Fred Hutchinson Cancer Research Center, Seattle, where she was awarded with a Cancer Research Institute Fellowship. Since March 2000 she holds a position of Project Leader in the Cancer Immunotherapy and Gene Therapy Program and in the Bone Marrow Transplantation Unit of HSR, and she teaches Hematology in the School of Medicine Università Vita e Salute, Milano. Since 2000 she is coordinating a European working group on Suicide Gene Therapy in Bone Marrow Transplantation. 55

56 Gene Therapy-Mediated Immune Evasion to Facilitate Islet Transplantation in Type 1 Diabetes Assoc. Prof. Dr. Ahter Dilsad Sanlioglu 1,2 Center for Gene and Cell Therapy 1 & Department of Medical Biology 2, Akdeniz University Faculty of Medicine, Antalya, Turkiye, Type 1 Diabetes (T1D) is characterized by an autoimmune destruction of the insulin-producing pancreatic beta cells. Although pancreatic islet transplantation has been a very promising approach for T1D treatment, its success is limited by islet graft malfunction and secondary graft failure. Thus, pretransplant ex vivo gene manipulation strategies of the islets emerged as a major area of research, utilizing many different molecules to increase the overall success of the islet transplantation. TNF-Related Apoptosis-Inducing Ligand (TRAIL) is a type II transmembrane protein that can interact with the transmembrane death receptors TRAIL-R1 (DR4) and TRAIL-R2 (DR5) which signal for apoptosis, and transmembrane decoy receptors TRAIL-R3 (DcR1) and TRAIL-R4 (DcR2), which are unable to transduce apoptotic signals [1, 2]. TRAIL is well known for its selective apoptotic effects on a wide range of cancer cells. Yet its exact role in the development of T1D remains to be clarified. TRAIL s potential role in T1D has been studied by two animal models of autoimmune diabetes [3]. In one model, blocking of the TRAIL function with a soluble TRAIL receptor was shown to accelerate diabetes in NOD mice, with an increased degree of autoimmune inflammation. In the second model, TRAIL-deficient C57BL/6 mice that received multiple doses of streptozotocin (STZ) displayed an earlier onset of diabetes along with a higher degree of islet inflammation, compared to the normal mice that were put through the same experimental procedures. In addition to these results suggesting an important role for TRAIL in down-regulation of the autoimmune inflammatory response in T1D, there are also results implicating a destructive role for TRAIL in T1D [4, 5]. We hypothesized that TRAIL overexpression in beta islets would be protective against the actions of the autoreactive cytotoxic T cells (CTL), the major actors in islet inflammation, and thus provide a prolonged survival of the transplanted islets in STZ-induced diabetic rats [6]. Diabetic rats carrying under their kidney capsules the pancreatic islet grafts transduced ex vivo with TRAILencoding adenoviral vectors (Ad5hTRAIL) displayed prolonged normoglycemia compared to those grafted with mock-infected or AdCMVLacZ-infected islets. In addition, severity of insulitis was also reduced in rats engrafted with Ad5hTRAIL-infected islets, which suggested a clear therapeutic benefit. TRAIL s possible role in suppressing the autoimmune inflammatory response in T1D development was also implicated by our group in a different experimental setting, where alterations in the expression levels of TRAIL ligand and receptors at different stages of T1D development were investigated in NOD mice treated with STZ or cyclophosphamide (CY). TRAIL expression was observed to be prominently decreased and kept at very low levels along the disease course selectively following cyclophosphamide treatment, which is well known for its suppressing effects on the T regulatory cells, thus easing development of the diabetic indications [7]. In conclusion, TRAIL ligand appears as a suitable molecule to be used to prolong the graft survival and thus increase the overall success in pancreatic islet transplantations, providing a successful immune evasion strategy through suppression of the inflammatory response when overexpressed in the islet grafts. 56

60 OP-1 EXPRESSION OF CHEMOKINE-LIKE RECEPTOR 1 (CMKLR1) ON MACROPHAGES CO- CULTURED WITH FIBROBLAST AND/OR TUMOR CELLS: MODELING THE INFLUENCE OF MICROENVIRONMENT Dorina RAMA, Basic Oncology, Oncology, Ankara Güneş ESENDAĞLI, Basic Oncology, Oncology, Ankara Dicle GÜÇ, Basic Oncology, Oncology, Ankara Aim of the study: Influence of microenviroment on the macrophage maturation or terminaldifferentiation is a well established phenomenon. On the other hand, maturation of macrophages can be impeded in the tumor microenvironment. CMKLR1, the receptor for chemerin, is expressed on tissue-resident mature macrophages (e.g. peritoneal macrophages) but not widely distributed on the circulating monocytes. We hypothesized that CMKLR1 expression is a simultaneous event during the differentiation of the tissue-resident macrophages and asked whether the tumor microenvironment affects the level of CMKLR1 expression. Methods: The presence of CMKLR1 was determined by using an in vitro macrophage co-culture model employing fibroblasts and tumor cells. The modulation of CMKLR1 was assessed on monocyte/macrophage cell line J744A.1. Flow cytometry, Reverse transcription-polymerase chain reaction (RT-PCR), Enzymelinked immunosorbent assay (ELISA), Methyl-thiazolyl-tetrazolium (MTT) assay, Immunocytochemistry and morphological analysis were performed. Results: CMKLR1 expression was found to be associated with the fibroblast-assisted maturation of J744A.1 monocyte/macrophage cells in the co-cultures established to model tumor microenvironment, whereas the presence of tumor cells was able to upregulate CMKLR1 expression independent of macrophage maturation. In addition, macrophages cultured with tumor cells or in tumor cellconditioned media responded to recombinant chemerin (17-156) peptide and increased the expression of proinflammatory IL-1β, TNF-α and IL-12 p40 cytokines. The native form of chemerin (prochemerin) supplied by fibroblasts did not induce a functional response. Conclusion: These observations may indicate a potential role for chemerin and CMKLR1 in the regulation of inflammatory responses in the tumor microenvironment. Keywords: macrophage, differentiation, CMKLR1, Chemerin, cancer, tumor microenvironment 60

63 OP-3 XRCC1 SUPPRESSES SOMATIC HYPERMUTATION AND PROMOTES ALTERNATIVE- NONHOMOLOGOUS END JOINING IN IGH GENES Huseyin SARIBASAK, Department of Basic Medical Sciences, Sifa University, Izmir Robert W. MAUL, Lab. of Molecular Bililogy and Immunology, National Institute on Aging, Baltimore, MD/USA Zheng CAO, Laboratory of Cancer Immunodiagnostics, Van Andel Research Institute, Grand Rapids, MI/USA William YANG, Lab. of Molecular Bililogy and Immunology, National Institute on Aging, Baltimore, MD/USA Daniel MCNEILL, Lab. of Molecular Gerontology, National Institute on Aging, Baltimore, MD/USA David M. WILSON, Lab. of Molecular Gerontology, National Institute on Aging, Baltimore, MD/USA Patricia J. GEARHART, Lab. of Molecular Biology and Immunology, National Institute on Aging, Baltimore, MD/USA Activation-induced deaminase (AID) deaminates cytosine to uracil in immunoglobulin genes. Uracils in DNA can be recognized by uracil DNA glycosylase and abasic endonuclease to produce single-strand breaks. The breaks are either repaired faithfully by DNA base excision repair (BER), or mutagenically to produce somatic hypermutation (SHM) and class switch recombination (CSR). To unravel the interplay between repair and mutagenesis, we decreased the level of a scaffold protein in BER, X-ray cross-complementing 1 (XRCC1). Using mice heterozygous for XRCC1, we observed a striking increase in the frequencies of SHM in variable regions from Peyer\'s patch cells, and of double-strand breaks in the switch regions during CSR. Although the frequency of CSR was normal in Xrcc1+/- splenic B cells, the length of microhomology at the switch junctions decreased, suggesting that XRCC1 also participates in alternative-nonhomologous end joining. Furthermore, haploinsufficient B cells had reduced Igh/c-myc translocations during CSR, supporting a role for XRCC1 in microhomology-mediated joining. Our results imply that AID initiates many single-strand breaks in variable and switch regions, which become substrates simultaneously for both BER and mutagenesis pathways. Keywords: XRCC1, Class Switch Recombination, Somatic Hypermutation, Alternative, End Joining, AID 63

64 OP-4 THE IDENTIFICATION OF THE TUMOR MICROENVIRONMENT MEMBERS, SPECIFICALLY CANCER ASSOCIATED FIBROBLASTS, FOR FUNCTIONAL ANALYSES IN A RAT CHEMICAL MAMMARY CARCINOM Gurcan GUNAYDIN, Basic Oncology, Hacettepe University Institute of Oncology, Ankara Yusuf DOLEN, Basic Oncology, Hacettepe University Institute of Oncology, Ankara Sacit Altug KESIKLI, Basic Oncology, Hacettepe University Institute of Oncology, Ankara Dicle GUC, Basic Oncology, Hacettepe University Institute of Oncology, Ankara Constituents of the tumor microenvironment are among key players determining the cancer cell behavior. Seed-centric models do not take into account the probable impact of the microenvironment, soil, that the tumor cells reside in. Fibroblasts are among the most common types of cells found both in connective tissues and the tumor microenvironment. They are thought to be involved in a dynamic crosstalk with other cells of the tumor microenvironment. The aim of this study is to characterize and effectively isolate cancer associated fibroblasts (CAFs) for further functional assays.n-nitroso-n-methyl Urea (NMU) induced experimental mammary carcinogenesis model was utilized. 21 days old 20 female Spraque-Dawley rats were injected once-a-week for four weeks with i.p. 50mg/kg NMU. For the control experiments, seven rats were injected with isotonic saline using the same protocol. When the animals are about two months old, developed tumors were harvested surgically under sterile conditions for CAF isolation. Fibroblasts were isolated from breast tumor tissues using a protocol which utilizes collagenase I and hyaluronidase. Enzymatically digested tissues were then cultured in fibroblast-selective-medium. The same protocol was also used to isolate normal tissue fibroblasts from healthy mammary tissues. These CAFs and healthy tissue fibroblasts were immunostained to show their differential expressions of surface markers such as α-smooth Muscle Actin (α-sma) and Vimentin, in order to distinguish CAFs from their normal tissue counterparts. These immunostainings clearly showed that CAFs had significantly higher levels of α-sma expression than normal fibroblasts. This finding of differential α-sma expression was shown to fade out with further passages of cultured CAFs. Histological examinations also revealed significant morphological differences between these cells. In conclusion, CAFs were successfully propagated using a rat chemical breast carcinogenesis model. Further functional analyses with these CAFs will be performed employing various coculture systems to investigate their roles in immunity against breast cancer. Keywords: cancer, associated fibroblast, breast cancer, tumor microenvironment, chemical carcinogenesis 64

65 EMPATHY- SYMPATHY, NAMELY TOLERANCE 65

66 OP-5 PRO-INFLAMMATORY AND REGULATORY PROPERTIES OF CASPASE-1 ARE DIFFERENTIALLY MEDIATED BY ITS SUBSTRATES IL-1BETA AND IL-18 IN HELICOBACTER INFECTION Ayça SAYI YAZGAN, Molecular Biology & Genetics, Istanbul Technical University, Istanbul Iris HITZLER, Inst. of Molecular Cancer Research, University of Zurich, Zurich Anne MÜLLER, Inst. of Molecular Cancer Research, University of Zurich, Zurich Activated pro-inflammatory cysteine protease caspase-1 processes pro-il-1beta and pro-il-18 to generate the bioactive cytokines and to initiate pathogen-specific immune responses. Little information is currently available on caspase-1- and inflammasome activation during infection with the gastric bacterial pathogen Helicobacter pylori. Our work have addressed a possible role of caspase-1 and its cytokine substrates in the spontaneous and vaccine-induced control of Helicobacter infection, and the development of gastritis and gastric cancer precursor lesions, using experimental infection and vaccine-induced protection disease models. We show caspase-1 activation and IL-1 signaling are absolutely required for the efficient control of Helicobacter infection in vaccinated mice. IL-1R-/- mice fail to develop protective immunity, but are protected against Helicobacter-associated gastritis and gastric preneoplasia. In contrast, IL-18 is dispensible for vaccine-induced protective immunity, but essential for preventing excessive gastric immunopathology. In conclusion, we show for the first time that the processing and release of a regulatory caspase-1 substrate, IL-18, counteracts the pro-inflammatory activities of another caspase-1 substrate, IL-1beta, thereby balancing control of the infection with the prevention of excessive gastric immunopathology. Keywords: Helicobacter pylori, caspase-1, innate immune recognition, IL-18 66

69 OP-7 IMMUNIZATION WITH A DNA CHIMERIC MOLECULE ENCODING A HEMAGGLUTININ PEPTIDE AND A SCFV CD21-SPECIFIC ANTIBODY FRAGMENT INDUCES STRONG LONG- LASTING CTL RESPONSE T Nikola KEREKOV, Immunology, Institute of Microbiology, Bulgarian Academy of Sciences, Sofia József PRECHL, Immunology, Immunology Research Group, Hungarian Academy of Sciences, Budapest Nikolina MIHAYLOVA, Immunology, Institute of Microbiology, Bulgarian Academy of Sciences, Sofia Nina IVANOVSKA, Immunology, Institute of Microbiology, Bulgarian Academy of Sciences, Sofia Andrey TCHORBANOV, Immunology, Institute of Microbiology, Bulgarian Academy of Sciences, Sofia Objectives: There are several important advantages of the DNA vaccines, using naked DNA encoding viral antigens over currently used protein vaccines, with the most significant being the fact that DNA vaccines has the potential to induce cytotoxic immune response as live attenuated viral vaccines do. We hypothesized that sequences encoding an epitope of influenza A virus hemagglutinin (IP) attached to sequences encoding a scfv antibody fragment against a costimulatory B cell surface receptor would result in the in vivo expression of a chimeric viral peptide with increased immunogenicity. Methods: We have inserted the DNA construct into pnut protein expression vector system. The constructs contained the Fab fragment of an antibody against mouse CR1/2 linked to IP peptide representing a conserved epitope of influenza A virus hemagglutinin and to the product of either the human oncogene products FOS or jun, having a natural tendency to bind each another. This would result in the forming of more stable bivalent chimeric IP-carrying scfv molecules Female Balb/C mice aged 8 to 10 week old were injected intramuscularly with a cocktail of 25µg of each construct per mouse. A complex immunization scheme was developed in which we apply one or combination of two types of vaccines. Antibodies against the considered epitope in the sera of injected animals were measured with ELISA. The cell-mediated immune response induced in the animals was evaluated using a cytotoxic assay. Results: The immunization with a plasmid containing the described construct induced a strong anti-influenza cytotoxic response lasting for more than 6 months. After prime-boosting with protein chimeric molecule we obtained anti-influenza cytotoxic and antibody response. Conclusion: Immunization of mice with pure DNA, encoding the antigen of interest attached to a scfv antibody fragment to mouse positive receptor, followed by prime-boosting has been successfully used to induce protective immunity against a model pathogen. Keywords: scfv fragments, chimeric molecule, influenza, virus DNA vaccine 69

70 70

71 OP-8 IMMUNE RESPONSE TO HEPATITIS B SURFACE ANTIGEN DISPLAYED ON PIII PROTEIN OF FILAMENTOUS BACTERIOPHAGE M13 Ibrahim HATIPOGLU, Vaccine Technology Laboratory, Genetic Engineering and Biotechnology Institute, Kocaeli B.Koray BALCIOGLU, Immunogenetics Laboratory, Genetic Engineering and Biotechnology Institute, Kocaeli Ibrahim SOGUT, Vaccine Technology Laboratory, Genetic Engineering and Biotechnology Institute, Kocaeli Aylin OZDEMIR BAHADIR, Immunogenetics Laboratory, Genetic Engineering and Biotechnology Institute, Kocaeli Serkan UZYOL, Immunogenetics Laboratory, Genetic Engineering and Biotechnology Institute, Kocaeli Deniz DURALI, Vaccine Technology Laboratory, Genetic Engineering and Biotechnology Institute, Kocaeli Hulya SIVAS, Anadolu University, Faculty of Science, Department of Biology, Eskisehir Berrin ERDAG, Immunogenetics Laboratory, Genetic Engineering and Biotechnology Institute, Kocaeli Aynur BASALP, Immunogenetics Laboratory, Genetic Engineering and Biotechnology Institute, Kocaeli Hepatitis B still remains one of the most common causes of death worldwide despite the presence of an effective vaccine for more than 20 years. Hepatitis B virus (HBV) vaccine is the first successful vaccine produced with genetic engineering technology and prepared with aluminum adjuvant. Although the aluminum compounds are still the most commonly used adjuvants in human vaccination, they mostly augment humoral immunity and not effective in stimulating cellular immunity. Phage particles are naturally immunostimulatory molecules inducing both humoral and cellular immunity and hence used as an alternative adjuvant in vaccine formulation. In the present study, Hepatitis B virus surface antigen (HBsAg) was expressed on piii protein of M13 phage and efficacy of recombinant phage was tested in mice. Briefly, HBsAg gene was amplified by PCR using a plasmid containing the whole HBV genome (ATCC, pam6) as a template. After cloning into pcantab5e phagemid vector and transforming into E. coli TG1 bacteria, positives clones were infected with M13 helper phages. DNA sequence of positive clones was analyzed with CEQ 8800 Sequence Analyzer. BALB/c mice were immunized with 109, 1010, 1011 and 1012 recombinant phage particles and the control groups were given wild type phage only. Results have shown that immunization with wild type phage elicited non-specific anti-hbsag response in BALB/c mice, unexpectedly. To evaluate its immunogenicity in mice having different genetic background, BALB/cj mice were then immunized with the same amount of recombinant phages. It was shown that immunization of BALB/cj mice with the highest dose of recombinant phage (1012/mice) was able to induce HBsAg-specific antibody response. Our results demonstrates the successful cloning of HBsAg on piii protein of M13 phage as a whole protein and testing its effect on immune response in different mice. Keywords: Hepatitis B Virus, Vaccine, Phage Display Method, adjuvant 71

72 NOVEL MOLECULAR TARGETS IN CLINICAL IMMUNOLOGY 72

73 OP-9 PRODUCTION AND CHARACTERIZATION OF POLYCLONAL ANTIBODY AGAINST FIBRONECTIN FOR ITS DETECTION ON THE SURFACE OF HUMAN SPERMATOZOA Forough TORABI, Faculty of Basic Sciences, Science and Research Branch, Islamic Azad University, Tehran Elham SAFARPOOR, Faculty of Basic Sciences, Science and Research Branch, Islamic Azad University, Tehran Niknam LAKPOUR, Nanobiotechnology Research Center, Avicenna Research Institute, ACECR, Tehran Mohammad Mehdi AKHONDI, Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR, Tehran Nazila AMINI, Faculty of Basic Sciences, Science and Research Branch, Islamic Azad University, Tehran Amir Hassan ZARNANI, Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR, Tehran Hamed HEIDARI VALA, Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR, Tehran Mohammad Reza SADEGHI, Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR, Tehran Introduction: Fibronectin (FN) is a 440 kda glycoprotein that influences a variety of cellular functions including: cell to cell adhesion, cell differentiation, survival and also sperm-oocyte binding. It s a component of spermatozoa and the basement membrane of the seminiferous tubule.due to the presence of FN on human spermatozoa and its role in fertilization process, this study was conducted to producea highly specific polyclonal antibody against FN in order to detect Fn on the surface spermatozoa. Methods: Human plasma FN was used to immunize a rabbit. Bleeding and antibody detection through ELISA assay was done on serum of rabbit. The produced antibody was purified by protein G affinity chromatography column. The quality and purity of the antibody was evaluated by SDS-PAGE. Its specific interaction and affinity of antibody was determined by ELISA. Immunoreactivity of the antibody with FN on human spermatozoa was evaluated through western blotting (WB), immunocytochemistry (ICC) and flow cytometry methods. Menstrual blood cells and HepG2 cell line were used as positive controls (Fig 1, 2). Results: The results showed that the antibody had desired purity and reactivity with the FN using SDS-PAGE and ELISA, respectively. It could also detect a band of about 440 kda corresponding to FN in spermatozoa by WB. Moreover, ICC analysis of human sperm showed that, the antibody enable to detect FN on the head of sperm. Also FN-positive sperm was % 12.45±10.38 of total spermatozoa by flow cytometry assay. Conclusion: FN most likely plays a role in sperm-oocyte interaction. This study shows that, the produced anti-fibronectin antibody can be used for detection of FN on spermatozoa surface. Keywords: Fibronectin, Spermatozoa, Fertilization, Flow cytometry 73

78 OP-12 NEW APPROACHES FOR SELECTIVE IMMUNOTHERAPY OF AUTOIMMUNE DISEASES BY ENGINEERED CHIMERIC MOLECULES Andrey TCHORBANOV, Immunology, Institute of Microbiology, Sofia Nikolina MIHAYLOVA, Immunology, Institute of Microbiology, Sofia Aim: In many physiological processes, peptides play a critical role as neurotransmitters, hormones, antibiotics. There are many approaches for immunotherapies using peptides: some of them are important components of chimeric molecules for immunosuppression and could be chemically or genetically engineered into defined conformational active form. The peptides contain epitopes, which are recognized by the immune system and protein-engineered or genetically engineered peptides conjugated to antibody-carrier could be used as a targeting device delivering the epitopes to the cells of interest. SLE is a complex autoimmune disease characterized by the generation of IgG auto-antibodies specific to nuclear antigens - dsdna, histones, nucleosomes. The appearance of pathological DNA-specific B cells in lupus is regarded as a major event in the development of the disease and their selective elimination is a legitimate goal in the efforts to control SLE. Inhibitory co-receptors such as FcγRIIb, CR1 and CD22 negatively regulate BCR signaling. These surface receptors on disease-associated B-lymphocytes are potential target molecules for therapeutic intervention. One of the mechanisms for B cell suppression is the crosslinking of the surface immunoglobulins with the inhibitory receptors by IgG-peptide-containing immune complexes. Methods: Protein engineering; Gene-engineering; Flow cytometry; Animal models Results: By protein and gene-engineering we constructed several chimeric molecules by coupling different self - mimicking peptides to an anti-mouse FcγRIIb or anti-human CR1-binding monoclonal antibody. The administration of these chimeric molecules to lupus-prone MRL/lpr mice or to humanized SCID mice transferred with PBMC from lupus patients resulted in the reduction of the levels of IgG anti-dsdna and Histone 1 antibodies, of the albuminuria levels, and in prevention of the development of glomerulonephritis. Conclusion: Protein- and gene-engineered chimeric molecules for targeting of antigens to the inhibitory receptors on human or mouse B cells are a tool for directed immune response modulation. 78

79 Fig. 1A Fig. 1B Fig. 2 79

80 POSTER PRESENTATIONS 80

81 MOLECULAR SENSORS OF THE INNATE IMMUNE SYSTEM 81

82 PP-1 THE EFFECT OF CXCL7-MEDIATED IMMUNE RESPONSES ON TUMOR BIOLOGY AND IMMUNOLOGY IN EXPERIMENTAL LUNG ADENOCARCINOMA MODEL Nese UNVER, Basic Oncology, Oncology, Ankara Gunes ESENDAGLI, Basic Oncology, Oncology, Ankara Dicle GUC, Basic Oncology, Oncology, Ankara Neutrophils are emerging as important mediators in cancer progression. The neutrophil chemoatractant protein, CXCL7 (NAP-2) has been identified at early stages of patients with lung cancer. Aim: The aim of this study is to evaluate the effect of CXCL7 transfected cancer cells on neutrophil functions and the role of this chemokine in the early and late anti-tumor immune responses in the experimental lung adenocarcinoma model. Methods: Neutrophils were cocultured with supernatants of control or CXCL7 modified LLC1 lung adenocarcinoma cells. ROS production, phagocytosis and chemotactic activities of neutrophils were evaluated. Control and CXCL7 modified LLC1 cells were inoculated into C57BL/6 mice. The tumor-infiltrating immune (Gr- 1, CXCR2, F4/80, CD4, CD8, CD25) cells were analysed by flow cytometry. In addition, ROS synthesis and phagocytosis of myeloid cells obtained from tumor tissue were evaluated. Neutrophil-associated chemokine and chemokine receptors were also analysed by Real Time PCR. Results: Supernatant of CXCL7 modified LLC1 cells increased the chemotaxis of neutrophils compared to the controls. ROS synthesis of neutrophils cultured with tumor supernatants was increased independent of CXCL7. Phagocytic activity of neutrophils cultured in the presence 10 ng/ml of recombinant CXCL7 was decreased. Macrophage infiltration in CXCL7-modified tumors was increased compared to the controls. During the early phases of immune response in CXCL7- modified tumors CD4+CD25+ population was decreased compared to the control tumors. The expression of CXCL3, CXCL5, CXCL7, CXCR1 genes were increased in CXCL7-modified tumors. Conclusion: CXCL7 overexpression can result in enhanced macrophage accumulation instead of expected neutrophil infiltration. These macrophages will be further characterized to understand the possible contributions of CXCL7 overexpression to tumor progression. 82

83 PP-2 INTERLEUKIN-33 DOES NOT ALTER NITRIC OXIDE PRODUCTION IN STIMULATED OR UNSTIMULATED MACROPHAGES Handan AKSOY, immunology, Gazi University, Ankara Vedat BULUT, immunology, Gazi University, Ankara Murine macrophages produce large amounts of nitric oxide (NO) on stimulation by interferon (IFN)-γ and lipopolysaccharide (LPS) or a high concentration of LPS alone. So far, induction or inhibition of NO production by many other cytokines, such as IL-4, IL-13, IL-10, TNF- and so on, have been reported. It was demonstrated that endothelium-derived NO played a critical role in promoting angiogenesis and vascular permeability induced by IL-33. However, the effect of IL-33 on NO production in macrophages has not yet been investigated. The gene encoding IL33 has been identified and classified as a new member of interleukin-1 family cytokines. Signalling of IL- 33 is transduced via ST2 receptor. Whereas IL-1 and IL-18 promote Th1 associated type I immune response, IL-33 induces Th2 polarisation. This study was aimed to explore the effect of IL-33 in macrophage cell line, J Therefore, IL-33 was added into cell cultures of stimulated or unstimulated J774 1 cells, by IFN- plus LPS or high dose LPS alone. Then, nitrite levels were measured by Griess reaction. We observed that IL-33 in a variety of concentrations did not lead to NO production in J774.1 cell line. IL-33 did not affect stimulated or unstimulated J774.1 cells by IFN- plus low dose LPS or high dose LPS alone in a dose dependent manner and in time-point experiments. On the other hand, we monitorized that stimulated macrophages with LPS or LPS plus IFN-γ could not persist in their viability. On contrary, IL-33 induced macrophages had longer life span. Macrophages can be polarised into various distinct subsets, classified as M1 and M2. NO production is a major sign for M1 phenotype. In conclusion, we suggest that IL-33 does not alter NO synthesis in J774.1 cell line with M1 characteristics. Keywords: Interleukin-33, macrophages, nitric oxide 83

84 PP-3 THE EFFECTS OF SYSTEMIC MESENCHYMAL STEM CELL TRANSPLANTATION ON WOUND HEALING OF ISCHEMIC COLONIC ANASTOMOSES Gökhan Tolga ADAŞ, Department of General Surgery, Okmeydani Education and Research Hospital, Istanbul Bahar ERYAŞAR, Department of Immunology, Institute for Experimental Medicine (DETAE), Istanbul Soykan ARIKAN, Department of General Surgery, Istanbul Education and Research Hospital, Istanbul Oğuzhan KARATEPE, Department of General Surgery, Sisli Etfal Education and Research Hospital, Istanbul Erdal KARAÖZ, Center for Stem Cell and Gene Therapies Research and Practice, Kocaeli University, Kocaeli Gülçin GACAR, Center for Stem Cell and Gene Therapies Research and Practice, Kocaeli University, Kocaeli Özgür KEMIK, Department of General Surgery, Yuzuncu Yil University, Medical Faculty, Van Gülçin KAMALI, Department of Patology, Okmeydani Education and Research Hospital, Istanbul Duran ÜSTEK, Department of Genetics, Institute for Experimental Medicine (DETAE), Istanbul OBJECTIVE: The goal of this study is to examine if allogenic mesenchymal stem cell (MSC) transplantation is a useful therapy for left ischemic colon anastomosis in rats. SUMMARY: Problems with anastomosis healing, especially anastomotic leakages, may lead to serious postoperative complications and increase morbidity and mortality. The colon is the most ischemiasensitive area in gastrointestinal tract and ischemia is one of the most important causes of colonic anastomosis leakage and wound dehiscence. Bone marrow-derived mesenchymal stem cells (BM-MSCs), which are also referred to as stromal progenitor cells, are self-renewing and expandable stem cells. Recent studies have suggested that BM-MSCs play a crucial role in the processes of intestinal repair and accelerate angiogenesis. MATERIAL- METHODS: BM- MSCs derived from rat femur and tibia were examined for phenotypical characterisation. Forty male Wistar albino rats weighing g were divided into four equal groups (n=10) as follows: group 1: control, ischemic left colonic anastomoses (sacrified on fourth day); group 2: control, ischemic left colonic anastomoses (sacrified on seventh day); group 3: ischemic left colonic anastomoses + systemic transplanted BM-MSCs (sacrified on fourth day); group 4: ischemic left colonic anastomoses + systemic transplanted BM-MSCs (sacrified on seventh day). Histopathological features and anastomotic strength were evaluated. RESULTS: BM-MSCs therapy significantly accelerated all of the healing parameters for ischemic colonic anastomosis except for inflammation and augmented the levels of the hydroxyproline and bursting pressure on the fourth day. Histological parameters, especially angiogenesis, were also found to be important for healing of ischemic colonic anastomoses. CONCLUSIONS: This is the first study to use systemic transplanted cell therapy for the healing of ischemic colonic anastomosis. BM-MSCs therapy significantly accelerated the healing parameters for ischemic colonic anastomosis. Keywords: Mesenchymal stem cells, Ischemic colonic anastomosis, Intestinal wound healing, Anastomotic dehiscence 84

86 PP-4 MACROPHAGE POLARISATION VIA PROLONGED LOW-DOSE LIPOPOLYSACHHARIDE INDUCTION Handan AKSOY, Immunology, Gazi University, Ankara Vedat BULUT, Immunology, Gazi University, Ankara We investigated those effects of prolonged low dose LPS induction in macrophage J774.1 cell line. For this aim, cells were repeatedly challenged with 10ng/ml LPS every three days for a period of 3 weeks. Control groups were not subjected to any stimulation. In our study, NO production was measured by Griess reaction and chemokine, CXCL10 (IP-10), was determined by ELISA. In order to observe chemokine receptors, such as CCR2 and CCR7, on stimulated and unstimulated cells, IFA staining was performed. It was seen that there was an elevated NO production of cells starting from first day and reached to upper level (66, 37+14,65 nmol / 106 cells) by make a peak in a week. When we compared with control groups and other scheduled groups, there was statistically significant difference (p<0,01). There was decrease to level of control group as from second week. In addition, getting increase chemokine IP10 (CXCL10) production of cells was observed as of first day (7509,40+44,92 pg/ml) and they stabilized this level during 4 days (7257,97+195,75 pg/ml). There were statistically significance in first and fourth day measurements when compared control group and other scheduled groups (p<0,01), and had fallen to level of control group after a week (178,53+2,25 pg/ml). Cells expressed CCR2 and CCR7 under high dose 1µg/ml LPS or IFN-γ 40U/ml and 10ng/ml LPS stimulation. These data showed that prolonged low dose LPS induction caused to skew toward M1 polarisation. These observations are important because the body systems are continuously exposed to the external environment via the air inhaled or bacterial colonization in mucosal field or skin which contains numerous potentially harmful agents. Lipopolysaccharide is a component of the gram-negative bacterial cell wall. Persistent exposure to LPS has caused to modulate immune system and plays a critical role in macrophage polarisation. Keywords: lipopolysaccharide, macrophages, polarization 86

87 PP-5 NANOLIPOSOMES ENCAPSULATING NUCLEIC ACID BASED TLR LIGANDS INDUCES INFLAMMASOME MEDIATED POTENT IMMUNE ACTIVATION Banu BAYYURT, Molecular Biology and Genetics, Science, Ankara İhsan GÜRSEL, Molecular Biology and Genetics, Science, Ankara AIM: Intracellular TLRs recognize nucleic acids of microbial world and initiates a proinflammatory or inflammatory immune cascade. Clinical applications of the TLR ligands are significantly hampered either due to pre-mature in vivo degradation via nucleases or rapid clearance via adsorption onto serum proteins. Liposome encapsulation is a promising approach to spare labile ligands` early digestion. In vivo stability and promotion of cell triggering is mediated by sustained release of liposomal cargo that make liposomes an attractive tool in applied immunology. We have generated different nanoliposomes with varying net surface charges and loaded them either with TLR3 or TLR9 ligands in order to study their immune stimulatory features. METHODS: Thioglycollated peritoneal exudate cells (PECs), bone marrow derived DCs, and mouse splenocytes were treated with different liposome formulations harboring CpG (A or B-types) and pic (TLR3 and TLR9 ligands, respectively). The cells were studied by FACS, while the supernatants were analyzed for cytokine production by ELISA. RESULTS: Our data revealed that when DCs were treated with liposomal formulations, DC maturation and co-stimulatory molecule expression of DCs were improved up 50 fold compared to free CpG or pic induced activation. The IL6, IL12 and IFNg production from splenocytes and DCs were significantly boosted by liposomes. This improvement was highest for CpG-A loaded anionic liposomes, whereas stealth cationic liposomes were the best formulation for CpG-B type. Neutral and stealth cationic liposomes were very effective formulations for pic mediated cytokine production and DC maturation. Surprisingly, cholesterol rich formulations were effective inflammasome activators as evidenced by IL1b production of stimulated PECs. DISCUSSION: Our data implicated for the first time that a liposome formulation rich in cholesterol and encapsulating nucleic acid TLRLs improved not only DC maturation but also Th1-cytokine secretions. This activation is largely dependent on IL1b mediated inflammasome activation by nanoliposomes. Keywords: Liposome, Inflammasome, TLR ligands, Th1 cytokine 87

92 PP-10 CPG LOADED FLOURESCENT POLYMERIC NANOPARTICLES: A THERANOSTIC DRUG DELIVERY SYSTEM SUITABLE FOR TLR BASED THERAPIES Tamer KAHRAMAN, Molecular Biology and Genetics, Bilkent University, Ankara Banu BAYYURT, Molecular Biology and Genetics, Bilkent University, Ankara Vusela IBRAHIMOVA, Chemistry, Bilkent University, Ankara Donus TUNCEL, Chemistry, Bilkent University, Ankara Ihsan GURSEL, Molecular Biology and Genetics, Bilkent University, Ankara AIM: Designing nanoparticulate delivery systems suitable for simultaneous imaging have been attracting great interest. Several systems such as liposomes, micelles, dendrimers, nanospheres and nanocapsules are potential theranostic carriers in biomedical applications. Here, for the first time we describe a complexation strategy allowing us to deliver a TLR ligand along with simultaneous imaging of the tissues with fluorescent polymeric nanoparticles (NPs). METHOD: Four different NPs (P1, P2, P3 and P4) were used for biocompatibility and drug delivery experiments. RAW264.7 cells were incubated with NPs for indicated time periods and analyzed by FACS and confocal microscopy. Cytotoxicity experiments were performed with Dojindo reagent on RAW264.7 cells. In addition, cellular uptake of NPs in the presence of several scavenger receptor ligands was also investigated. Next, NPs were incubated with CpG ODN to yield nanocomplexes. In vitro and in vivo immunostimulatory effect of nanocomplexes was analyzed both on PBMCs or splenocytes. Pro-inflammatory cytokine production was assessed either by ELISA or in some cases by PCR. RESULTS: FACS and confocal analyses revealed that NP internalization kinetics was polymer dependent. Cytotoxicity experiments demonstrated that up to 100µg/ml concentrations none of the four NPs were toxic. Cellular uptake of amphiphilic particles are partly mediated by scavenger receptors (SRs), dextran sulfate pre-treated cells, a ligand for SRs, blocked >60% of NPs internalization. IL-6 and IP10 ELISA showed that NP1-nanocomplexes substantially improved CpGODN activity, furthermore, it significantly increased expression of TNFα, MIP1α, MIP1β and several other TLR transcript expressions upon ip. injection of mice. CONCLUSION: We demonstrated that fluorescent polymeric nanoparticles are well tolerated and can readily form nanocomplexes with nucleic acid ligands. NPs are promising candidates not only for cell tracking and imaging applications but at the same time capable of triggering immune activation via its cargo. Our findings suggest that these materials offer great potential for theranostic applications. 92

93 NOVEL DISCOVERIES IN THE ADAPTIVE IMMUNE SYSTEM 93

94 PP-11 EFFECT OF SHORT DURATION INTENSIVE EXERCISE ON MITOCHONDRIAL SUPEROXIDE DISMUTASE GENE EXPRESSION AND ANTIOXIDANT ACTIVITY IN YOUNG ACTIVE WOMEN Bakhtiar TARTIBIAN, Department of Exercise Physiology, Urmia University, Urmia Behrouz BAGHAIEE, Department of Exercise Physiology, Urmia University, Urmia Behzad BARADARAN, Department of Medical Science, Tabriz Medical Science University, Tabriz Noushin AZADPOUR, Department of Exercise Physiology, School of Sport Sciences and Technology, Hacettepe University, Ankara Amir MONFAREDAN, Department of Exercise Physiology, Tabriz Azad Islamic University, Tabriz Aim: During intense exercise, high levels of free radicals are produced, which may result in oxidative stress. But, regular exercise training enhances antioxidant defense system and protects against exercise-induced free radical damage. There have been few studies concerning the relationship between exercise and human manganese superoxide dismutase (MnSOD) gene expression. Therefore, the purpose of this study was to investigate the effect of short duration intensive exercise on mitochondrial MnSOD gene expression and total antioxidant capacity (TAC) in young active women. Methods: Fifteen young active women (aged years) were volunteered for this study. Venous blood samples were taken in three stages, before exercise, immediately after and 3 hours after exercise (time: 30 minutes, grade: 6 %, speed: 12 km/h). Real time PCR method was used for analysis of the MnSOD gene expression and the spectrophotometry method was used for measurement of TAC. A repeated measure design was used for this study. Results: Results showed no significant increase on the mrna levels of MnSOD in immediately and 3 hours after exercise (P1>0.421, P2>0.209). But significant increase was observed in the TAC levels only in immediately after exercise (P<0.031). Conclusion: Short duration intensive exercise does not significantly affect on MnSOD mrna level in active women, because they are adapted to free radicals and thus have high levels of antioxidant enzyme activity. However, further studies are needed to investigate the role of exercise on the MnSOD gene expression and antioxidant enzyme activity in young active women. Keywords: MnSOD, Gene expression, Active women 94

95 PP-12 DYNAMICS OF HUMAN TH17 DIFFERENTIATION FROM PERIPHERAL BLOOD NAÏVE CD4 T CELLS Semih Can AKINCILAR, MBG, İzmir Institute of Technology, İzmir Bünyamin AKGÜL, MBG, İzmir Institute of Technology, İzmir Ayten NALBANT, MBG, İzmir Institute of Technology, İzmir Naïve CD4 T helper cells can differentiate into IL-17 producing T cells, known as Th17 cells. The differentiation, maintenance and effector function of human IL-17 producing CD4 T cells are under intensive investigation. The aim of the study is to analyze the kinetics of human Th17 cell differentiation and to understand the dynamics of Th1, Treg and Th17 conversion under Th17 polarizing conditions. Human naive CD4 T cells were differentiated in vitro and the signature cytokine and transcription factor of Th subsets were analyzed. Data showed that activated CD4 T cells became IL17 expressing T cells. IL17 expressing CD4 T cells were also expressing RORC2 which is a specific transcription factor that drives Th17 differentiation. Interestingly, there were also IL17 expressing RORC2 negative CD4+T cells. The IFNγ and Foxp3 expressing cells were also detected. Furthermore, there were IL17/IFNγ, IL17/Foxp3 and RORC2/T-bet intermediate cells. Under Th17 polarizing conditions naïve CD4 T cells polarized into Th17 cells, but there was a considerable level of Th1 and Treg differentiation. The presence of Th1/Th17 and/or Th17/Treg intermediate is indicative of plasticity or trans-differentiation of Th subsets. A better understanding of Th differentiation may provide novel therapeutic opportunities in treating immunopathologies. 95

96 PP-13 SERUM AND MUCOSAL IMMUNOGLOBULINS BLOCK THE AUTOREACTIVE HUMAN IGG Andrey TCHORBANOV, Immunology, Stefan Angelov Institute of Microbiology, Sofia Iglika DJOUMERSKA-ALEXIEVA, Immunology, Stefan Angelov Institute of Microbiology, Sofia Iliyan MANOYLOV, Immunology, Stefan Angelov Institute of Microbiology, Sofia Purpose: About 20 % of the B-cells of the immune system of healthy individuals synthesize IgG antibodies, recognizing a great number of self antigens. The natural IgG autoantibodies are found in human serum and as an outcome - in intravenous immunoglobulin preparations (IVIg). Their function is to recognize the autoantigens some of which are also target of pathologic autoantibodies. A great enhancement of the natural IgG autoantibody activity was observed after treatment of IgG with dissociating agents (buffers with low or high ph value used in affinity column elution), but not after the use of non-denaturizing methods for antibody purification. Methods: The in vitro interactions between human colostral proteins or human serum proteins (both from healthy individuals) or pooled human IVIg and human liver antigens were investigated. Based on two different methods of isolating (the one using low ph treatment of antibodies, the other not) pure IgA, IgM were obtained from the different fractions of serum and mucosal proteins (distribution based on their molecular mass). Results: Inhibition of the interaction between affinity column isolated IgG or pooled IVIg preparations, on one hand, and human liver antigens, on the other, by purified serum or mucosal IgM, IgA and the F(ab )2 fragments was observed. Conclusion: The results allow us to conclude that the binding of auto-reactive IgG can be suppressed by serum or mucosal IgA and IgM. Our study supports the theory that IgM has a role in blocking the IgG autoantibodies. We suggest that normal pooled human IgM preparations have a positive immunoregulatory activity in patients with autoimmune diseases, as the auto-reactivity control is highly inefficient in those cases. Keywords: natural autoantibodies, IVIg 96

97 PP-14 THE EFFECTS OF EARLY BASAL INSULIN TREATMENT ON INFLAMMATION AND ENDOTHELIUM-RELATED BIOMARKERS IN PATIENTS WITH TYPE 2 DIABETES S. Altug KESIKLI, Department of Basic Oncology, Hacettepe University Institute of Oncology, Ankara Ugur UNLUTURK, Department of Endocrinology and Metabolism, Ankara University Faculty of Medicine, Ankara Gurcan GUNAYDIN, Department of Basic Oncology, Hacettepe University Institute of Oncology, Ankara Can ATES, Department of Biostatistics, Ankara University Faculty of Medicine, Ankara Dicle GUC, Department of BAsic Oncology, Hacettepe University Institute of Oncology, Ankara Ali Riza UYSAL, Department of Endocrinology and Metabolism, Ankara University Faculty of Medicine, Ankara Rifat EMRAL, Department of Endocrinology and Metabolism, Ankara University Faculty of Medicine, Ankara Background: In type 2 diabetes mellitus (T2DM), there is a lack of comparison of different treatment alternatives, especially in terms of pleiotropic effects such as the effects on endothelial functions, coagulation status and inflammation. Aim: This study was mainly prepared to analyze the effects of early basal insulin versus sulfonylurea treatment on endothelial injury, coagulation status and inflammation-related biomarkers in patients with T2DM. Methods: A total of 43 patients with T2DM, 16 in the insulin Detemir treatment arm and 27 in the gliclazide-mr arm were included. The study protocol is summarized in Figure 1. Plasma levels of soluble markers of endothelial activation or damage, such as intercellular cell adhesion molecule-1 (sicam-1), vascular cell adhesion molecule-1, E-selectin and P-selectin; markers of inflammation like monocyte chemoattractant protein-1 and interleukin-6, and markers to determine the tendency towards coagulation, like tissue plasminogen activator and plasminogen activator inhibitor-1 levels were investigated using commercially available ELISA kits. Results: Both treatment protocols were equally effective in controlling blood glucose. sicam-1 levels were observed to decrease significantly between the basal and 6th month follow-up measurements in the early insulin treatment arm. Moreover, se-selectin levels were demonstrated to decrease between the basal and 3rd month follow-up measurements only in the insulin Detemir arm. Although sp-selectin levels were demonstrated to increase significantly in both patient groups, the increment in the insulin Detemir arm was significant only between the 3rd and 6th month follow-up measurements, showing the effect of the addition of sulfonylurea to the treatment protocol. Levels of other markers were indifferent between the two treatment groups. Conclusion: These results suggest that basal insulin treatment might be a promising approach in preventing late complications associated with T2DM, through inhibiting endothelial activation and damage. This study was supported by The Scientific and Technological Research Council of Turkey (TÜBİTAK), Grant No: 110S119 Keywords: Type 2 Diabetes Mellitus, Endothelium, Insulin Detemir, Gliclazide-Modified Release, Inflammation 97

98 98

99 PP-15 INTRACELLULAR IFN-GAMMA AND IL17 DOUBLE POSITIVE CELLS IN SSPE PATIENTS* Durmus BURGUCU, Physiology, Akdeniz University School of Medicine, Antalya Dilara Fatma KOCACIK UYGUN, Pediatric Immunology-Allergy, Akdeniz University School of Medicine, Antalya Serkan FILIZ, Pediatric Immunology-Allergy, Akdeniz University School of Medicine, Antalya Nilgun SALLAKCI, Pediatric Immunology-Allergy, Akdeniz University School of Medicine, Antalya Sadi KOKSOY, Mikrobiology, Akdeniz University School of Medicine, Antalya Senay HASPOLAT, Pediatric Neurology, Akdeniz University School of Medicine, Antalya Ugur YAVUZER, Physiology, Akdenzi University School of Medicine, Antalya Olcay YEGIN, Pediatric Immunology-Allergy, Akdenzi University School of Medicine, Antalya Subacute Sclerosing Panencephalitis (SSPE) is a chronic, progressive inflammatory disease of central nervous system which is characterized by demyelinisation, astrogliosis, lymphocyte, macrophage and plasma cell accumulation around the blood vessels. Consistent abnormalities or defects in IL-12-Th1-G-IFN and/or IL-4-Th2-IL-4,13 axis have not been reported yet. A recently described subtype of helper T cells, namely Th17 cells ie IL-17 and IL-22 secreting cells, have been found to play important roles in experimental allergic encephalomyelitis (EAE) in mice and in multiple sclerosis in humans. We studied the IL17 and IFN gamma containing cells in patients with SSPE and healty controls. Intracellular cytokine measurements were performed by flow cytometry. It was found that the number of IL17 containing cells were higher than controls in SSPE patients (p:0.04). In addition, when stimulated with measles peptides, the IL17 containing cells from SSPE patients increased in number with respect to control cells. Moreover, number of both IL17 and IFNgamma containing cells were found to be higher than controls in SSPE patients (1.6% and 5.5% respectively,p: 0.04). In conclusion, our data reveals that in SSPE patients both the IL 12/IFN gamma axis and the IL 23/IL 17/IL 22 axis are responsive. The presence of double positive cells demonstrating both Th1 and Th17 features, it is possible to suggest that both Th1 and Th17 are involved in pathogenesis of SSPE. * This study is supported by Turkish Scientific and Technical Research Foundation (SBAG 109S089) 99

100 Double positive cells persentage of patients 100

101 PP-16 IL 17 RESPONSE IN SSPE PATIENTS* Dilara Fatma KOCACIK UYGUN, Pediatric Immunology-Allergy, Akdeniz University School of Medicine, Antalya Serkan FILIZ, Pediatric Immunology-Allergy, Akdeniz University School of Medicine, Antalya Nilufer CICEK EKICI, Pediatric Immunology-Allergy, Akdeniz University School of Medicine, Antalya Nilgun SALLAKCI, Pediatric Immunology-Allergy, Akdeniz University School of Medicine, Antalya Durmus BURGUCU, Physiology, Akdeniz University School of Medicine, Antalya Senay HASPOLAT, Pediatric Neurology, Akdenzi University School of Medicine, Antalya Ugur YAVUZER, Physiology, Akdenzi University School of Medicine, Antalya Olcay YEGIN, Pediatric Immunology-Allergy, Akdeniz University School of Medicine, Antalya Subacute Sclerosing Panencephalitis (SSPE) is a chronic, progressive, fatal inflammatory disease of central nervous system which is characterized by demyelinisation, astrogliosis, lymphocyte, macrophage and plasma cell accumulation around the blood vessels. The immunopathogenesis of these slow virus infections have not been clarified yet. We investigated the IL17 mrna in these patients. Mononuclear cells obtained from SSPE patients and healthy controls were induced with measles peptides and RNA was isolated. Using real time RT-PCR, mrna levels of IL-12/23 p40, IL 23 p19 and IL 17 were determined. It was found that cells from SSPE patients exhibited higher levels of p40 (p: 0.044) and p19 (p: 0.02) mrna with respect to cells obtained from healthy people. IL17 mrna levels were higher than the control group but this difference did not reach to significance (p: 0.07) Our data demonstrated that in SSPE patients the IL 12/IFN gamma axis and the IL 23/IL 17 is responsive. *This study is supported by Turkish Scientific and Technical Research Foundation (SBAG 109S089) RT-PCR results after measles stimulation 101

102 PP-17 TLR-2-ACTIVATED B-CELLS SUPPRESS HELICOBACTER-INDUCED PRENEOPLASTIC GASTRIC IMMUNOPATHOLOGY BY INDUCING T REGULATORY-1 CELLS Ayca SAYI YAZGAN, Istanbul Technical University, Department of molecular Biology and Genetics, Istanbul Anne MÜLLER, University of Zurich, Institute of Molecular Cancer Research, Zurich B-cells regulate autoimmune pathologies and chronic inflammatory conditions such as autoimmune encephalomyelitis and inflammatory bowel disease. The potential counter-regulatory role of B-cells in balancing pathogen-specific immune responses and excessive immunopathology is much less understood due to the lack of appropriate persistent infection models. We show here that B-cells have the ability to negatively regulate adaptive immune responses to bacterial pathogens. Using mouse models of infection with Helicobacter felis, a close relative of the human gastrointestinal pathogen H. pylori, we found that B-cells activated by Helicobacter TLR-2 ligands induce IL-10-producing CD4 + CD25 + T regulatory-1 (Tr-1)-like cells in vitro and in vivo. Tr-1 conversion depends on TCR signalling and a direct T-/B-interaction through CD40/CD40L and CD80/CD28. B-cell-induced Tr-1 cells acquire suppressive activity in vitro and suppress excessive gastric Helicobacter-associated immunopathology in vivo. Adoptive co-transfer of MyD88- proficient B-cells and Tr-1 cells restores a normal gastric mucosal architecture in MyD88 -/- and IL- 10 -/- mice in a manner that depends on T-cellular, but not B-cellular IL-10 production. Our findings describe a novel mechanism of B-cell dependent Tr-1 cell generation and function in a clinically relevant disease model. In conclusion, we demonstrate here that the B-cell/Tr-1 cell axis is essential for balancing the control of Helicobacter infection with the prevention of excessive Th1- driven gastric immunopathology, promoting gastric mucosal homeostasis on the one hand and facilitating Helicobacter persistence on the other. Keywords: Regulatory B cells, Tr-1 cells, Helicobacter, TLR-2 ligand 102

104 PP-19 CYTOKINE SECRETION OF T HELPER CELL SUBSETS; DIFFERENT ASPECTS OF BEHÇET S DISEASE Esin ÇETİN AKTAŞ, Department of Immunology, Institute for Experimental Medicine (DETAE), Istanbul Fulya COŞAN, Department of Immunology, Institute for Experimental Medicine (DETAE), Istanbul Fatma GEÇGEL, Department of Internal Medicine, Division of Rheumatology, Kocaeli Zeliha EMRENCE, Department of Genetics, Institute for Experimental Medicine (DETAE), Istanbul Nilgun AKDENİZ, Department of Immunology, Institute for Experimental Medicine (DETAE), Istanbul Ayse CEFLE, Department of Internal Medicine, Division of Rheumatology, Kocaeli Gunnur DENİZ, Department of Immunology, The Institute for Experimental Medicine (DETAE), Istanbul Aim: Behçet s disease (BD) is a systemic inflammatory disease with recurrent inflammatory mucosal ulcerations, uveitis, arthritis and vascular involvement. Although innate immune system plays the major role in the pathogenesis, previous data has shown that some Th1 associated cytokines have role in the inflammation of BD. The CD4+ Th cells can be differentiated into Th1, Th2, Th17, Th22 and regulatory T (Treg) cells and secrete different cytokines to regulate immune system. In this study cytokine secretion of T helper cells in BD were investigated. Methods: The study group consists of BD patients that have not received any immunosuppressive treatment (n=26, mean age=37.2 ± 9.7) with mucocutaneous involvement and healthy subjects (n=12, mean age=32.5 ± 8). Lymphocyte subpopulations in peripheral blood samples and intracellular cytokine secretion including IL-5, IL-10, IL-17, IL-22 and IFN- of CD4+ T and FOXP3+ Treg cells were determined by flow cytometry. Mann-Whitney U test was used for non parametric values. Results: Although the percentages of IL-5 secreting Th2 and IL-17A+IFN- - Th17 cells were found similar in both groups, IL-17A-IL-22-IFN- + Th1 and IL-17A-IL-22+IFN- - Th22 cells are significantly increased in BD patients compared to healthy subjects (p=0.002 and p=0.028, respectively). There was also no significant difference for the expression of CD4, CD8, CD3, CD19, however percentages of Treg (CD4+CD25+FOXP3+) cells were dramatically reduced in BD (p=0.000) patients. Conclusion: These findings revealed that increased levels of IFN- secreting Th1 cells as well as Th22 cells might be associated with inflammatory reactions. Although elevated production of IL-17 secreting T cells have been reported, we did not find any significant difference. It has been shown that inflammatory IL-22 secreting cells are distinct from Th17 cells and Th1 cytokines inhibit IL-17 but not IL-22. In the same manner, increased levels of IFN- might suppress the IL-17 secretion. 104

105 EMPATHY - SYMPATHY, NAMELY TOLERANCE 105

106 PP-20 THE RELATIONSHIP BETWEEN THE SOLUBLE HLA-G LEVELS IN MATERNAL SERUM AND PREGNANCY OUTCOMES İsmail BIYIK, Obstetrics and Gynecology, Duzce University School of Medicine, Duzce İsmail ÖZDEMIR, Obstetrics and Gynecology Department, Duzce University School of Medicine, Duzce Mustafa ALBAYRAK, Obstetrics and Gynecology, Duzce University School of Medicine, Duzce Ahmet KARATAŞ, Obstetrics and Gynecology, Duzce University School of Medicine, Duzce Seyit Ali KÖSE, Obstetrics and Gynecology, Duzce University School of Medicine, Duzce Fatih KESKIN, Obstetrics and Gynecology, Duzce University School of Medicine, Duzce Introduction and Objective: Preeclampsia, intrauterine growth retardation, oligohydramnios, abortus, preterm birth and preterm rupture of membranes are significant pregnancy complications. İnsufficient trophoblastic invasion is play important role of the pregnancy complications s pathophysiology that said above. Soluble HLA-G1/G5 is a molecule that associated with trophoblast invasion. The studies about prediction of pregnancy complications are made. When pregnancy complications predict early, essential strategies improved and prevention will be come in to question. Aim of our study was investigate relationship first and second soluble HLA-G1/G5 levels, PAPP-A, free beta hcg, AFP, second trimester uterin artery doppler parameters with pregnancy complications. Materials and Methods: In our prospective study 180 pregnant followed up. In our study in addition to the pregnants that applicant for week down syndrome screening PAPP-A, free beta hcg, NT values, maternal blood soluble HLA-G1/G5 levels recorded. Pregnants AFP levels during week and uterine artery doppler parameters during recorded. In our study the relationship stated parameters with pregnancy complications are investigated. In addition evaluation of predictive values of markers, we compared the markers relation. The pregnants were classificated as normal pregnancy, preeclampsia, oligohydramios, IUGR, abortus, preterm birth, PROM 7 different groups. Results: First and second trimester HLA- G1/G5 levels was not significantly different between groups. But in preeclamptic patients first trimester HLA-G1/G5 levels and PAPP-A levels was significantly significantly lower than nonpreeclamptic pregnants. Conclusions: If pregnancy complications predicted in early pregnancy, high risk pregnants follow up closely. In this wise pregnancy complications will ve diagnose early, necessary preventative methods will be possible. But large series that suppport of our study is needed. Keywords: soluble HLA-G, first trimester, high risk pregnancy 106

107 First and second trimester biochemical parameters of groups 107

108 PP-21 IL-1BETA BREAKS IMMUNOTHERAPY-INDUCED T CELL TOLERANCE IN ALLERGIC ASTHMA PATIENTS Umut Can KUCUKSEZER, Dept. of Immunology, Istanbul University, Institute of Experimental Medicine, Istanbul Suzan ADIN-CINAR, Dept. of Immunology, Istanbul University, Institute of Experimental Medicine, Istanbul Bilun GEMICIOGLU, Dept. of Chest Diseases, Istanbul University, Cerrahpasa Faculty of Medicine, Istanbul Cezmi A. AKDIS, Dept. of Immunology, Swiss Institute of Allergy and Asthma Research (SIAF), Davos Mubeccel AKDIS, Dept. of Dermatology, Swiss Institute of Allergy and Asthma Research (SIAF), Davos Gunnur DENIZ, Dept. of Immunology, Istanbul University, Institute of Experimental Medicine, Istanbul Asthma is a disease of the respiratory system, with three main subgroups, the allergic group being the biggest proportion. Allergic asthma is cured by allergen-specific immunotherapy with success. Innate inflammatory cytokines and ligands for certain Toll Like Receptors (TLRs) are shown to release T-cell unresponsiveness to allergens in PBMCs of healthy individuals, in vitro. This study aims to investigate roles of these inflammatory factors in immunotherapy model which provides specific allergen-induced unresponsiveness in allergic asthmatic patients. PBMCs from three patients who received immunotherapy to known allergens were stained with CFSE and cultured with the existence and absence of immunotherapy allergen, and stimulated with IL-1beta, IL-6 and ligands for TLR4 and TLR8. First results of this ongoing study supports the tolerance-breaking effects of IL-1beta and IL-6, however more number of patients should be investigated. Keywords: Asthma, Allergen-specific Tolerance, Toll Like Receptors, Inflammation, Allergen 108

109 IMMUNOGENETICS IN HEALTH & DISEASE 109

110 PP-22 HLA-G POLYMORPHISMS ASSOCIATED WITH MOTHER-TO-CHILD HIV TRANSMISSION Roberta SANCHES, General and Specialized Nursing, University of São Paulo, Ribeirão Preto Francielly MATOS, General and Specialized Nursing, University of São Paulo, Ribeirão Preto Luciana TSUDA, General and Specialized Nursing, University of São Paulo, Ribeirão Preto Mariana SANTIAGO, General and Specialized Nursing, University of São Paulo, Ribeirão Preto Silvana QUINTANA, Gynaecology and Obstetrics, University of São Paulo, Ribeirão Preto Eduardo DONADI, Medical Clinic, University of São Paulo, Ribeirão Preto Ana Paula FERNANDES, General and Specialized Nursing, University of São Paulo, Ribeirão Preto HLA-G, a non classical MHC class I molecule, is highly expressed at maternal-fetal interface and is strongly involved in the regulation of maternal-fetal immune response. Studies have reported that HLA-G inhibits NK cells-mediated lysis and the immunosuppressive properties of HLA-G might contribute to the susceptibility and persistence of viral infections. The HLA-G 3 UTR polymorphisms have been associated with the insertion (I) or deletion (D) of a 14pb fragment, which may be involved with stability and expression levels of HLA-G mrna. The aim of this study was to analyse the hypothesis that HLA-G 3 UTR polymorphisms can influence the HIV vertical transmission. Blood samples were obtained from 51 mother-child pairs. The samples were drawn from two groups. For 19 mother-child pairs it was confirmed that both were HIV infected, and 32 mother-child pairs only mother was infected and the child was uninfected. Genomic DNA was extracted from blood samples in order to proceed the HLA-G 3 UTR genotyping using sequence specific probe analysis. Statistical analysis was performed using the Fisher exact test. We did not detect significance difference in allele and genotype frequencies between HIV-infected and HIVuninfected child. However, the I (insertion) allele was less frequent among exposed infected child (39%) as compared with HIV exposed uninfected child (52%). In addition, the I/I genotype was less frequent among exposed infected child (16%) as compared with HIV exposed uninfected child (25%). Our results suggest that HLA-G 14 pb deletion/insertion polymorphism may be associated with the HIV vertical transmission. 110

111 PP-23 MITOGENIC INTERVENTIONS AND THE FUNCTION OF T AND B LYMPHOCYTES IN RESTRAINT AND STREPTOZOCIN-INDUCED ZINC-TREATED DIABETIC MICE Musaad ALDUBAIB, Veterinary Medicine, Qassim University, Buridah Zinc is an essential trace element important for the efficiency of the immune system in reason of its widespread role in the activity of enzymes, transcription factors and cytokines. Numerous data indicates that zinc affects B and T lymphocyte activation, proliferation and lymphocyte-mediated cytolysis during diabetes and stress. To test this hypothesis, the present study was carried out to identify and quantify the modulating activities of zinc in vivo and in vitro using mitogenesis of lipopolysaccharide (LPS Sigma- USA), a specific B-cell mitogen and phytohaemagglutinin (PHA, Sigma-Aldrich, USA) a specific T-cell mitogen of peripheral lymphocyte. In vivo splenic and in vitro peripheral lymphocytes samples of restraint and streptozocin-induced mice diabetes were isolated by Ficoll-paque density centrifugation. The percentages of T and B cells activation with diabetes (32.15 ± 4.3% and ± 1.9 % P < 0.001) and with stress (23.65 ± 2.67% and ± 2.67% P < 0 05) were increased when compared with that of normal (11-7±3.01% and 15.56± 1.63%). In contrast, levels of T cells activated by phytohaemagglutinin-p in vivo did not differ from normal animals. Data obtained revealed that impaired lymphocyte function and enhanced susceptibility to infections is a common feature of mice diabetes and stress. Our results suggest that the zinc alteration is involved in the stress and streptozocin-induced mice diabetes. In conclusion, these results indicate that action of zinc on lymphocyte function in diabetes and stress may be important target for modulation of immune responses and understanding of mechanisms leading to several pathologies of immune cells observed in stress and streptozocin-induced mice diabetes. 111

112 PP-24 CIRCULATING SOLUBLE TUMOR NECROSIS FACTOR RELATED APOPTOSIS INDUCING- LIGAND (TRAIL) IS DECREASED IN TYPE-2 NEWLY DIAGNOSED, NON-DRUG USING DIABETIC PATIENTS Atil BISGIN, Department of Medical Genetics, Human Gene and Cell Therapy Center of Akdeniz University Hospital and Clinics, Antalya Arzu D. YALCIN, Department of Internal Medicine, Allergy and Clinical Immunology Unit, Antalya Education and Research Hospital, Antalya Reginald M. GORCZYNSKI, Division of Cellular & Molecular Biology, Toronto Hospital, University Health Network, Toronto We examined the association between serum soluble tumor necrosis factor (TNF)-related apoptosis-inducing ligand (strail) measured by ELISA and HbA1C levels, pre/post-prandial blood glucose levels and body mass index were evaluated as a surrogate marker of diabetes in 22 newly diagnosed type 2 diabetic patients without any other symptoms of complications. Concurrently, the levels of strail were examined in 14 healthy age and sex-matched individuals. TRAIL was originally isolated as an inducer of apoptosis and was an important component of the immune system. Although it is well acknowledged that it also has an important role in Type 1 (T1D) and Type 2 Diabetes (T2D) development, it is unknown whether TRAIL can inversely reflect the progression of diabetes from its early stage. There was no significant association between strail and HbA1C levels (ρ= 0.099, p=0.066) or post-prandial blood glucose level (ρ=0.025, p=0.91) or BMI (ρ=0.36, p=0.098). However, significant difference in strail levels was found between newly diagnosed non-drug using DM patients and control groups. In conclusion, to the best of our knowledge, this is the first study to assess serum strail levels in newly diagnosed type-2 diabetes mellitus patients without any complication and to evaluate the protein\'s relationship with clinical status. Our study demonstrates that strail levels are significantly lower in type-2 diabetes than the healthy controls. Further studies are needed to investigate whether the TRAIL system has a causal role or the role of strail as a marker in type-2 diabetes mellitus. Keywords: soluble TRAIL, type-2 diabetes mellitus, non-drug using diabetic patients Table 1: There is no correlation between BMI, blood glucose strail & HbA1C strail & Pre/Post-Prandial strail & BMI Level Blood Glucose Number of XY Pairs / Spearman r -0, ,03080 / 0, , % confidence interval to / to to to P value 0,6614 0,7615 / 0,9125 0,0978 Is the correlation significant? No (alpha=0.05) No No 112

113 PP-25 THE EFFECT OF HELICOBACTER PYLORI VIRULENCE FACTORS ON BAFF EXPRESSION IN GASTRIC EPITHELIAL CELLS Miray KARAYILAN, Faculty of Science & Letters, Molecular Biology & Genetics, Istanbul Nesteren MANSUR, Faculty of Science & Letters, Molecular Biology & Genetics, Istanbul Emre SOFYALI, Faculty of Science & Letters, Molecular Biology & Genetics, Istanbul Ayca SAYI YAZGAN, Faculty of Science & Letters, Molecular Biology & Genetics, Istanbul Helicobacter pylori has been identified as one of the main risk factors for development of gastric cancer. 80 % of population in developing countries are infected with H. pylori. 20 % of this patients develop one or more of gastric complications, such as gastric ulcers, gastric cancer and gastric B cell lymphoma. H. pylori-driven inflammation in stomach leads to follicular gastritis in childhood and MALT lymphoma in adults. These two types of pathology are characterized by the presence of B cell follicles and later on their transformation that leads to disruption of the tissue. To investigate how H. pylori-infected gastric epithelial cells affect local B cell activation may help to understand the mechanism of this pathology formation. Recently, it has been reported that B cell activating cytokine (BAFF) which is responsible for B cell maturation and survival, can be produced by H. pylori-infected gastric epithelial cells. However, the mechanism of BAFF expression through H. pylori infection is still unknown. The presence of H. pylori s Cag-PAI pathogenecity island is correlated with the severity of gastric malignancies. As, there is no evidence about the possible effect of Cag-PAI on BAFF production from gastric epithelial cells, we aim to investigate BAFF production from gastric epithelial cells (MKN45 cells) using H. pylori G27 strain and its deltapai (absence of the cag-pai pathogenecity island) mutant. Our preliminary data sugested that live H. pylori is not necessary for BAFF expression as H. pylori sonicate and to a lower extent its counterpart H. felissonicate can induce BAFF expression in MKN45 cells. Furthermore, BAFF expression in deltapai sonicate-treated cells are slightly lower than wild type sonicate. Taken together, our preliminary results suggest that B cell activation and maturation, are partially dependent on the cag PAI status of H. pylori. 113

118 PP-29 CLONING OF ATYPICAL CHEMOKINE RECEPTORS CRAM-A AND CRAM-B FOR COMPARATIVE FUNCTIONAL ANALYSIS Parisa SARMADI, Basic Oncology, Oncology, Ankara Gunes ESENDAGLI, Basic Oncology, Oncology, Ankara Atypical chemokine receptors play an important role in the termination of inflammatory responses. Upon binding to their cognate ligands, chemokine gradient drops and immune cell migration is hampered. CCRL2 is the newest member of atypical chemokine receptor family. The human CCRL2 gene has two transcript variants CRAM-A and -B. The aim of this study is to compare the effect of these different variants on the chemokine gradient. Total cdna was synthesized using RT-PCR. CRAM-A and -B protein coding cdnas, specific primers and Pfu DNA polymerase were used in amplification and site-directed cloning (NheI and XmaI restriction enzyme sites). CRAM-A and -B amplicons, pires2-egfp and pcdna3.1/ct-gfp eukaryotic expression vector were digested, isolated, ligated, and then transformed into E.coli competent bacteria. The resulting constructs were confirmed by restriction analysis and DNA sequencing. The recombinant clones pcram-a-ires2-egfp and pcram-b-ires2-egfp enabled the bicistronic expression of CRAM and EGFP, whereas pcdna3.1/cram-a-ct-gfp and pcdna3.1/cram-b-ct-gfp produced a hybrid GFP-tagged protein. The recombinant plasmids and empty vectors were delivered into HEK293 cell line via liposomal transfection. Transfection efficiency (GFP expression) and recombinant CRAM expression were analyzed by flow cytometry. In conclusion, the transcript variants of human CCRL2 gene were cloned into different recombinant DNA constructs and de novo expression of recombinant CRAM proteins were determined. Next, the ligand binding assays will be performed with CRAM-A or -B-expressing cells. Keywords: Atypical Chemokine Receptors, CRAM, Chemokine, Recombinant DNA Technology 118

119 PP-30 ANALYSIS OF AID MRNA LEVELS IN CLL PATIENTS WITH DIFFERENT CYTOGENETIC STATUS Metin Yusuf GELMEZ, Immunology, DETAE, Istanbul Ayşegül Başak AKADAM TEKER, Hematology, Istanbul Faculty of Medicine, Istanbul Aynur DAĞLAR ADAY, Hematology, Istanbul Faculty of Medicine, Istanbul Akif Selim YAVUZ, Hematology, Istanbul Faculty of Medicine, Istanbul Teoman SOYSAL, Hematology, Cerrahpasa Faculty of Medicine, Istanbul Günnur DENIZ, Immunology, DETAE, Istanbul Melih AKTAN, Hematology, Istanbul Faculty of Medicine, Istanbul The clinical course of CLL is highly variable and difficult to predict. The patients having deletion in 13q14, 17p13 or 11q22 have worse clinical outcome than patients with del13q34. While treatment for early-stage patients isn t required, the rest should be treated. Some of early-stage patients progress and will need treatment. It s important to detect which of these patients have poor prognosis and when to start treatment. Staging system cannot provide the critical information about which patients will develop progressive disease. AID causes point-mutations in IgV region to compose SHM and break-points in Ig switch region to compose CSR. Recent studies indicate that AID over-expression can cause point-mutations and translocations in non-ig genes. In this study, we aimed to analyze the AID expression in CLL patients with or without deletion in 17p13, 13q14, 11q22.3 or 13q34. METHODS: Peripheral blood was collected from 44 patients and 50 controls. Total RNA was isolated from blood using RNA Isolation Kit. cdna synthesis was done by using cdna Synthesis Kit. AID and a reference gene HPRT1 were analyzed by real-time PCR by using a LightCycler 480 (Roche). RESULTS: We didn t find significant differences in AID expression between CLL patients and controls. There were also no significant differences in AID expressions in patients without deletions compared to controls. We found AID expressions were significantly increased in patients with deletions. We also found AID expressions were significantly increased in patients with del17p or del13q14. AID expression was found to be in the border of statistical significance in patients with del11q. We didn t find significant differences in patients with del13q34. CONCLUSİON: Our results may suggest that AID over-expression can be associated with the disease progression and might be responsible for deletions in patients with CLL. AID can be useful clinical parameter, after its importance has been shown in larger and multivariate studies. Keywords: Activation induced cytidine deaminase, AID, CLL 119

120 Comparative Analysis Results 120

121 PP T/A AND GLY82SER POLYMORPHISMS IN RECEPTOR OF AGE (RAGE) EFFECT SOLUBLE RAGE LEVELS Esra BIRBEN, Pediatric Asthma and Allergy Unit, Hacettepe University, School of Medicine, Ankara, Turkey Cansın SACKESEN, Pediatric Asthma and Allergy Unit, Hacettepe University, School of Medicine, Ankara, Turkey Omer KALAYCI, Pediatric Asthma and Allergy Unit, Hacettepe University, School of Medicine, Ankara, Turkey Aim: Oxidation of amino groups of proteins, lipids and nucleic acids by the oxidant attack results in the production of Advanced Glycated End-products (AGE), whichfurther amplify the existing oxidative burden and augment inflammation. RAGE, well known AGE receptor, is the multi-ligand member of the immunoglobulin family. Two polymorphisms, Gly82Ser located in the ligand binding region and -374T/A is located in the promoter region may significantly influence the function of RAGE. We aimed to investigate the effects of -374T/A and Gly82Ser polymorphisms on plasma levels of soluble (s)rage and asthma phenotypes. Methods: 362 asthmatic children and 134 healthy controls were genotyped in both loci by PCR-RFLP analysis. Plasma RAGE levels were determined by ELISA in a randomly selected group of patients (n=83) and controls (n=31). Results: There was no difference in the frequency of genotypes or plasma levels of srage between asthmatic and healthy children. However, plasma levels of srage were significantly lower in both healthy and asthmatic children carrying the serine allele compared to those carrying the glycine allele (healthy children: 1433 pg/ml in glycine allele carriers vs 825 pg/ml in serine carriers; asthmatic children: 1540 pg/ml in glycine allele carriers vs 1117 pg/ml in serine carriers) (p<0.001). Similarly lower plasma srage levels were observed in T allele carriers of -374 polymorphism comparing to carriers of A allele in both co-dominant (p=0.027) and T dominant model in asthmatics (1411 pg/ml in TT and TA carriers and 1737 pg/ml in AA carriers) (p=0.011). Allergic phenotypes including atopy, eosinophil numbers, total IgE, FEV1% and FEV1/FVC ratio were not associated with the studied polymorphisms. Conclusion: Serine allele at Gly82Ser and T allele at -374 position are associated with lower srage levels in plasma. The functional consequences of this observation on markers of inflammation remain to be determined. Keywords: Asthma, polymorphism, RAGE, srage 121

123 PP-33 SERUM PROINFLAMATORY CYTOKINE LEVELS IN PANDEMIC H1N1 INFLUENZA A VIRUS INFECTION Cemal BULUT, Infectious Diseases and Clinical Microbiology, Ankara Training and Research Hospital, Ankara Zeliha KOCAK TUFAN, Infectious Diseases & Clinical Microbiology, Ankara Training & Research Hospital, Ankara Server YAGCI, Infectious Diseases & Clinical Microbiology, Ankara Training & Research Hospital, Ankara Cigdem ATAMAN HATIPOGLU, Infectious Diseases & Clinical Microbiology, Ankara Training & Research Hospital, Ankara Sami KINIKLI, Infectious Diseases & Clinical Microbiology, Ankara Training & Research Hospital, Ankara Ali Pekcan DEMIROZ, Infectious Diseases & Clinical Microbiology, Ankara Training & Research Hospital, Ankara Aim: We evaluated four proinflamatory cytokine levels in hospitalized patients with pandemic influenza H1N1, and correlate the results with the clinical aspects. Methods: A total of 27 patients admitted to our clinic for pandemic influenza H1N between 1st November 2009 and 31st January 2010 were included into the study. Serum samples of patients were drawn in the first and the last day of hospitalization. Epidemiological, clinical and laboratory findings of patients were retrieved from medical records retrospectively. Serum levels of TNF-alfa, IL-6, IL-10 and INFgamma were determined by ELISA method (Biosource,Vienna, Austria) according to the manufacturer s instructions. A total of 30 healthy volunteers were selected as control group. The Mann-Whitney U test was used for nonparametric variables. The Wilcoxon test was used to compare two paired groups. The association between nonparametric variables was determined by the Spearman correlation coefficient (r). Any value of P < 0.05 was considered as statistically significant. Results: On univariate analysis IL-10, IL-6 and INF-gamma levels were found to be significantly higher in the patient group (p<0,005) (Table). Between first and last day of the hospitalization, an increase was noted on IL-6, IL-10 and INF-gamma levels (p>0.05). The most seen symptoms were cough, fever and myalgia. Mean fever was 38.2±0.8ºC. A positive correlation was found between fever and TNF-alfa; IL-6 and nausea; serum LDH and CK levels; and negative correlation was found between TNF-alfa and serum GGT and LDH levels, and IL-6 and serum creatinin level (p<0.05) Conclusion: IL 6, IL 10 and INF gamma levels were found to be higher in H1N1 influenza patients with respect to healthy subjects. So we can assume that these three cytokine may have a role on the symptomatic phase of the disease. Keywords: proinflamatory cytokine levels, influenza, clinical symptoms, ELISA Table. Cytokine levels in patients and control group Patient group n=27 (pg/ml, mean±se) Control group n=30 (pg/ml, mean±se) IL-6 9.5± ±0.6 IL ± ±0.4 TNF alfa 11.6± ±2.1 INF gamma 34.6± ±

124 PP-34 ASSOCIATION BETWEEN HLA ALLELES AND JAK2 V617F MUTATION IN POLYCYTHEMIA VERA Fusun OZMEN, Department of Basic Oncology, Hacettepe University Institute of Oncology, Ankara Gozde GOREN, Department of Basic Oncology, Hacettepe University Institute of Oncology, Ankara Dicle GUC, Department of Basic Oncology, Hacettepe University Institute of Oncology, Ankara Emin KANSU, Department of Basic Oncology, Hacettepe University Institute of Oncology, Ankara Polycythemia vera (PV) is one of the myeloproliferative disorders (MPD) and a clonal disease of hematopoietic precursor cell. PV has a risk of disease progression into acute leukemia or myelofibrosis. JAK2 V617F mutation is very important for PV occurence that present in almost all PV patients (96-100%). This mutation contributes to abnormal myeloproliferation and progenitor cell hypersensitivity. Human Leukocyte Antigen (HLA) system is also releated to some hematological disease such as myelodisplastic sendrom, pre-b ALL and multiple myeloma. Present study aims to analyse association of HLA A,-B,-DRB1 alleles with JAK2 V617F mutation positive polycythemia vera disease. HLA allelles frequency was investigated in 75 PV patients with JAK2 V617F mutation in Turkey. Control population was created from 100 healthy donors who were registered to tissue typing laboratory for hematopoietic stem cell-transplantation. HLA genotyping was performed using PCR-SSP method at low-resolution level. Chi-square and Fisher s exact test were used for statistical evaluation. JAK2 V617F mutation was harbored by all of PV patients and HLA allleles analyzed in both the control and PV patients. Sixty different HLA alleles were identified in two grups. Although the certain HLA alleles frequency in the patient population including A*02(19,33%), A*03(18,66%), B*38(10%), DRB1*04(20%) were higher than the control population and only B*38 was significantly higher in PV patients (2,5% in control grup, p=0.003). HLA-A*74 alleles was one of the lowest frequency alleles in patients but it was differed significantly from the controls (1,33%, p=0.007). Certain HLA alleles were also found to be higher in the control population as compared to the patients including A*11(10,5%), B*35(21%), B*51(13,5%), DRB1*01(11%), DRB1*07(12%). In those, A*11 and DRB1*01 were statisticaly significant in control grup (respectively, p=0.001 and p=0.008). This results suggest that certain HLA alleles might be positively and negatively associated with JAK2 V617F mutation in polycythemia vera. Keywords: Polycythemia vera, HLA, JAK2 V617F mutation The statistically significant HLA allele frequencies in PV HLA-A* 11 HLA-A*74 HLA-B*38 HLA-DRB1*01 Frequency in Patients (%) 1,33 1, ,33 Frequency in Controls (%) 10,5 0 2,5 11 P value 0,001 0,007 0,003 0,

125 PP-35 PRODUCTION OF MONOCLONAL ANTIBODIES SPECIFIC FOR ACRA3 TOXIN OF ANDROCTONUS CRASSICAUDA SCORPION VENOM Figen CALISKAN, Biology, Eskisehir Osmangazi University, Eskisehir Emel ERGENE, Biology, Anadolu University, Eskisehir Hulya SIVAS, Biology, Anadolu University, Eskisehir İbrahim SOGUT, Genetic Engineering and Biotechnology, TUBITAK Marmara Research Center, Gebze-Kocaeli Ibrahim HATIPOGLU, Genetic Engineering and Biotechnology, TUBITAK Marmara Research Center, Gebze-Kocaeli Aynur BASALP, Genetic Engineering and Biotechnology, TUBITAK Marmara Research Center, Gebze-Kocaeli Scorpion stings are a serious health problem in many countries of the world. The venoms contain rich sources of different classes of toxic polypeptides that represent pharmacological tools for biological research. These peptides affect the ion-channel function of excitable and non- excitable cells and are known to be responsible for the neurotoxic effects. Four different families of toxins have been described, which specifically interact with ion channels: Na+, K+, Cl and Ca2+. Androctonus crassicauda is a widely distributed species of scorpion in the Middle East, including Turkey (Southern Anatolia), Iran, Iraq (Mosul), Syria (Palmyra, Homs, Damascus), Jordan and Saudi Arabia. A. crassicauda are known to be most medically important scorpion species of Turkey and cause severe intoxication in humans and thus requiring anti-serum therapy. Peptide toxin Acra3 consist of 66 amino acids residues with a molecular mass of 7,620.3 Da has been purified from Turkish scorpion A.crassicauda crude venom (GenBank ID: GQ454796). In this study, Acra3 toxin was purified from A.crassicauda scorpion venom by high performance liquid chromatography. BALB/c mice were immunized three times with Acra3 antigen and after boosting, the mouse with high antibody titer was selected for fusion. Lymphocytes were obtained from the lymph nodes and the spleen of mice and fused with F0 myeloma cells (ATCC CRL 1646) in the presence of polyethylene glycol. After successful fusion, 5B9 monoclonal antibody (MAb) specific for Acra3 antigen was developed and the cross reactivity of monoclonal antibody was tested with ELISA. 5B9 MAb production was performed in large scale, purified with S300 size exclusion chromatography and the immunoglobulin type was found to be IgM. The results showed that anti- Acra3 MAb 5B9 could be a bioactive tools for exploring the structure/function relationship and the pharmacological mechanism of scorpion peptide neurotoxins. Acknowledgments: This study was granted by TUBITAK TBAG-106T527 project. Keywords: Androctonus crassicauda scorpion venom, monoclonal antibody, neutralization 125

126 PP-36 INVESTIGATION OF THE SDF-1 AND CXCR-4 GENE POLYMORPHISMS RELATIONSHIP WITH TYPE 2 DIABETES MELLITUS Burcin AYDIN, Department of Immunology, DETAE, Istanbul Ender M. COŞKUNPINAR, Department of Molecular Medicine, DETAE, Istanbul Sema BİLGİÇ GAZİOĞLU, Department of Immunology, DETAE, Istanbul Bedia ÇAKMAKOĞLU, Department of Molecular Medicine, DETAE, Istanbul Gunnur DENİZ, Department of Immunology, DETAE, istanbul M. Temel YILMAZ, Department of Internal Medicine, Istanbul Medical Faculty, Istanbul Ali Osman GÜROL, Department of Immunology, DETAE, Istanbul Purpose Type 2 diabetes mellitus (T2DM) is one of the leading causes of death and disability in the world. Also it accounts approximately 90% of all diabetic cases. Chemokines may play an important role in the development of T2DM. The goal of this study is to analyze the chemokines such as stromal cell-derived factor-1 (SDF-1) and chemokine receptor type 4 (CXCR-4). SDF-1 is a small peptidic chemokine that regulates many essential biological processes such as migration of progenitor cells, and CXCR-4 plays an important role in the mobilization of cells from the bone marrow. The polymorphism of these two chemokines in patients with T2DM were investigated and compared with healthy subjects. Method Seventy-five patients with T2DM and 75 healthy subjects were enrolled in the study. DNA was isolated from heperinized periferic blood samples. Real-time PCR assay was carried out using LightSnip (rs Roche-Germany) for SDF-1 and LightSnip (rs Roche-Germany) for CXCR-4. Results Compared to healthy subjects, T2DM patients were found significantly different in terms of age, BMI (body mass index), gender and smoking status (p<0.05 age, p<0.05 gender, p=0.007 BMI, p=0.012 smoking). The frequencies of the TT, TC and CC for SDF-1 exon 5 were 0%, 18.7%, 81.3% in T2DM patients and 0%, 15.1%, 84.9% in healthy subjects, respectively. Frequencies of CXCR-4 exon 3 genotypes were 60% AA, 32% AT, 8% TT in T2DM patients and 71.2% AA, 26.0% AT, 2.7% TT in healthy individuals. There was 1.6-fold increased risk in T2DM patients compared to healthy subjects. Conclusion Although an increased risk was found in CXCR-4 exon 3 TT genotype carrying individuals, SDF-1 and CXCR-4 polymorphisms may not have a role in susceptibility to T2DM. Further studies with an increased number of subjects are needed in this field. Keywords: type 2 diabetes, sdf-1, cxcr-4, polymorphisms 126

128 PP-38 INCREASED SERUM STRAIL LEVEL IN NEWLY DIAGNOSED STAGE-IV LUNG ADENOCARCINOMA BUT NOT SQUAMOUS CELL CARCINOMA, IS CORRELATED WITH AGE AND SMOKING Atil BISGIN, Division of Oncology, Department of Clinical and Experimental Medicine, University of Linköping, Linköping, Sweden Aysegul KARGI, Division of Medical Oncology, Department of Internal Medicine, Antalya Education and Research Hospital, Antalya Arzu D YALCIN, Allergy and Clinical Immunology Unit, Department of Internal Medicine, Antalya Education and Research Hospital, Antalya Saadet GUMUSLU, Department of Biochemistry, Faculty of Medicine, Akdeniz University, Antalya Bulent KARGI, Department of Thoracic Surgery, Medical Park Hospital, Antalya Reginald M GORCYZNSKI, Division of Cellular & Molecular Biology, Toronto Hospital, University Health Network, Toronto, Canada Background: Lung cancer is the leading cause of cancer mortality in the world. Many factors can protect against or facilitate the development of the cancer. A TNF family member TRAIL, has a complex physiological role more than merely activating the apoptotic pathway in cancer cells. Furthermore, vitamin D is converted to its active form locally in the lung, suggesting that it may play an important role in lung health. With the prospect of more effective therapeutic options for advanced stage disease, there are still insufficient current follow-up procedures for lung cancer. Aim: Clinical significance of serum strail and 1,25-dihydroxyvitamin D(3) level in patients with non-small cell lung cancer was investigated. Methods: Consecutive 18 adenocarcinoma and 22 squamous cell carcinoma patients of non-small cell lung cancer referred to our institute were included in this study. There were 12 men and 6 women, ages ranged from 38 to 97 years, with adenocarcinoma and average of 60.5 years. And there were 20 men and 2 women, ages ranged from 46 to 80 years, with squamous cell carcinoma and average of years. Curative resection was performed for all patients. Serum levels of strail and 1,25-dihydroxyvitamin D(3) were measured in the samples of time of diagnosis. Results: Circulating strail levels of NSCLC patients were significantly higher than the control group. Although there was no correlation between patient survival and strail levels, the high strail levels in adenocarcinoma patients were correlated with age and cigarette smoking. Interestingly, the strail level of healthy individuals was correlated with serum 1,25-dihydroxyvitamin D(3). Conclusions: Serum strail concentrations changed significantly during NSCLC perpetuation and correlated with age and smoking in adenocarcinoma. However, it seems that the concentration of this protein has no critical value as a prognostic factor and no effect on survival rate in NSCLC. Keywords: soluble TRAIL, non-small cell lung cancer, adenocarcinoma, squamous cell carcinoma, smoking 128

129 Figure 1: Scatter dot plots. 129

130 PP-39 POLYMORPHISM OF HLA-G 3 UNTRANSLATED REGION IN HIV/HCV COINFECTION Luciana TSUDA, General and Specialized Nursing, University of São Paulo, RIBEIRÃO PRETO Fernando VILAR, Faculty of Medicine of Ribeirão Preto, University of São Paulo, RIBEIRÃO PRETO Alcione MACHADO, Faculty of Medicine of Ribeirão Preto, University of São Paulo, RIBEIRÃO PRETO Ana MARTINELLI, Faculty of Medicine of Ribeirão Preto, University of São Paulo, RIBEIRÃO PRETO Eduardo DOANADI, Faculty of Medicine of Ribeirão Preto, University of São Paulo, RIBEIRÃO PRETO Ana Paula FERNANDES, General and Specialized Nursing, University of São Paulo, RIBEIRÃO PRETO Background: The HLA-G molecule may perform various functions depending on the situation in which it is expressed, and has been associated with persistence of viral infection. HLA-G 3 untranslated region polymorphisms are related to their levels of expression. Furthermore, the identification of HCV genotype is clinically important because it allows the establishment of the duration of therapy and prognosis of infection. Methods: 50 HCV infected patients and 50 HIV/HCV coinfected patients in outpatient treatment were recruited for the study. HLA-G 3 untranslated region was typified and evaluated by direct sequencing of the PCR-amplified product. Genotypes of HCV were obtained from medical records. This study was approved by the local ethics committee. Results: In HIV/HCV group, 15 subjects (30%) were homozygous for deletion, 5 (10%) homozygous for insertion and 30 (60%) heterozygous for insertion/deletion. In comparison with the haplotypes of the HCV group, 21 subjects (42%) were homozigous for deletion, 7 (14%) homozigous for insertion and 22 (44%) heterozygous for insertion/deletion (P = , and , respectively, by Fisher\'s exact test). Regarding the HCV genotype in HIV/HCV group: the 1 was observed in 40 (80%) patients; 3 in 5 (10%); 1a/1b in 4 (8%) and 2 in 1 (2%) patient. HCV genotype in HCV group: the 1 was obeserved in 26 (52%) patients, 3 in 20 (40%); 1a/1b in 3 (6%) and 2a/2c in 1 (2%) patient. Conclusions: Although any statistically significant difference was found, in both groups were prevalence of heterozygous insertion/deletion to the HLA-G 3\'untranslated region. With respect to HCV genotypes, the 1 prevailed in both coinfected and monoinfected groups. Keywords: HIV, HCV, HLA-G polymorphism, AIDS 130

131 PP-40 THE INVESTIGATION OF SUPPRESSORS OF CYTOKINE SIGNALLING (SOCS-1 & SOCS-3) GENE EXPRESSION IN SICKLE CELL ANEMIA Hatice SEVGİ, Biology, Science, Mersin Ahmet Ata ÖZÇİMEN, Biology, Science, Mersin Ozlem ÖZDEMİR, Pediatrics, Medical Biology, Mersin Figen AYMAK, Immunology, Medical Biology, Bursa Gorkem UMUT, Medical Biology, Bursa YAasemin KAÇAR, Biology, Science, Mersin Selma ÜNAL, Pediatrics, Medical Biology, Mersin Haluk Barbaros ORAL, Immunology, Medical Biology, Bursa OBJECTIVE: The sickle cell anemia (SCA) is a disorder which caused by a point mutation and results from a single aminoacid subsititution of valine for glutamic acid at the sixth position of the beta-globin chain. SOCS-1 and SOCS-3 genes are two members of suppressor of cytokine signaling genes. In this study, suppressors of cytokine signaling in the pathogenesis of SCA are an inflammatory disease of gene expression level for the first time investigated. SOCS-1 and SOCS-3 gene expression levels were evaluated according to stages (in the pre-crisis and crisis) of the disease. METHODS: The blood samples were collected from SCA patients and healthy groups and lymphocytes were isolated and managed cdna synthesis after purification of total RNA. Amplification was carried out by quantitative TaqMan real-time PCR technique using specific primers and probes to Socs-1 and Socs-3 genes. Beta-actin gene was used for normalization of the genes. RESULTS: The patients with crisis, sickle cell anemia and healthy controls were compared in terms of SOCS-1 and SOCS-3 gene expression levels. SOCS-1 gene expression in the crisis group, was significantly lower than thosed of sickle cell anemia and healthy controls but Socs-3 expression did not change compared to the control group. CONCLUSIONS: SOCS-1 gene expression decreased during the crisis. These data suggest that SOCS-1 gene seems to associated with inflammation in SCA and may make a positive impact of reducing inflammation. 131

133 PP-42 THE EVALUATION OF ANA AND DSDNA RESULTS Engin KARAKECE, Microbiology Laboratory, Sakarya University Educational and Research Hospital, Sakarya Ihsan Hakki CIFTCI, Microbiology Laboratory, Sakarya University Educational and Research Hospital, Sakarya Ali Rıza ATASOY, Microbiology Laboratory, Sakarya University Educational and Research Hospital, Sakarya Detection of auto-antibody by Enzyme Linked Immunosorbent Assay (ELISA) technique is used as the method for diagnosis of autoimmune disease. This study was aimed to compare the measurement of antinuclear antibodies (ANA) and double-stranded DNA (ds DNA) by ELISA in serum samples. Serum samples were obtained from two sources: 1) sent to the laboratory for ANA testing, and 2) sent to the laboratory for ds DNA. Each of these sera was tested for the presence of ANA and dsdna by ELISA kits. This tests were performed by commercial Aeskulisa dsdna check (AESKU DIAGNOSTICS, Germany) according to the manufacturer's instructions. ANA and ds DNA results were classified as positive or negative for each patient. Borderline results were arbitrarily classified as positive. For the patients; 2.96 % (58/1975) were ANA positive and 4.52 % (29/642) were ds DNA positive by ELISA. There was no significant correlation between ANA and ds DNA positivity. Only two patients ELISA kit results were found positive regarding for both ANA and ds DNA. Agreement between assays was generally marginal. There are number of factors which may contribute to the variability between these assays. The ELISA ANA assay, in our laboratory, uses a commercial kit utilizing an IgG-specifik second antibody. One might expect that this decrease the frequency of false-positive results, but this was not the case with all of the ELISA kits. Hence, each of the assay provides different criteria for clinicians and ANA screen test should be followed up with a fluorescent test to provide information on ANA pattern. Keywords: autoimmunity, ANA, dsdna 133

134 PP-43 PRESENCE AND CONFIRMATION OF DONOR SPECIFIC ANTI-HLA ANTIBODIES IN PATIENTS WHO WILL RECEIVE SOLID ORGAN Ali SENGUL, Immunology, Medical Park Hospital, Antalya Mehtap ULKER DEMIREL, Immunology, Medical Park Hospital, Antalya Ayse Tuba OZTEKIN, Immunology, Medical Park Hospital, Antalya Aysegul KILINCAY, Immunology, Medical Park Hospital, Antalya Emine CAPKIN COLAK, Immunology, Medical Park Hospital, Antalya Kamil DOGRU, Immunology, Medical Park Hospital, Antalya Objective: Sensitization is an important barrier for solid organ transplantation. The presence of circulating antibodies to donor antigens is detected by various lymphocyte cross-match (LCM) test techniques. The complement dependent cytotoxicity (CDC) methods may result false positivity. In this study we aimed to determine the true -positive results. Methods:1409 patients who will receive kidney and pancreas transplants between November 2008 and the end of 2010 were tested for CDC-LCM. 71 Patients with positive T and/or B LCM results by CDC method were re-evaluated by flow-cytometric (FC) LCM and/or panel reactive antibody (PRA) class-1 and class-2 tests. 85 Patients with positive T and/or B LCM results by CDC method were re-evaluated only by PRA class-1 and class-2 tests. Results: Totally 1707 CDC-LCM tests performed for 1409 patients with various donors. Out of 1707 tests, 1318 (77.21%) had negative and 389 (22.79%) had positive results. Out of 389 positive donor-recipient pairs 71pairs were re-evaluated by FC-LCM: 17 (23.94%) had both B and T CDC-LCM positivity and only 11 confirmed by FC-LCM; 2 (2.81%) had only T CDC-LCM positivity and found as negative by FC-LCM; 52 (73.24%) had only B CDC-LCM positivity and only 15 confirmed by FC-LCM. Discussion:A prospective pre-transplant donorrecipient crossmatch test is performed to confirm the presence or absence of donor HLA specific alloantibodies. Positive crossmatch in a sensitised patient is a contraindication to transplantation. CDC-LCM test based on cell death by antigen, antibody and complement complex reaction. Any factor, which is resulted cell death will give the positive test result. This study suggests that it is possible to determine the true or false positive CDC-LCM test results by FC-LCM. Keywords: Lymphocyte cross-match, Donor specific antibody, Transplantation 134

135 PP-44 THE COMBINATORY EFFECT OF KCNH2/HERG1 K897T AND DIO2 THR92ALA IN THE SUSCEPTIBILITY TO SCHIZOPHRENIA Adil COLAK, Bioengineering, Chemical and Metalurgical Engineering Faculty, Istanbul Gokce AKAN, Medical Biology and Genetics, The Instute of Health Science, Istanbul Fatih ONCU, Forensic Psychiatry Unit, Bakırköy Psychiatric and Neurological Diseases Hospital, Istanbul Dogan YESILBURSA, Forensic Psychiatry Unit, Bakırköy Psychiatric and Neurological Diseases Hospital, Istanbul Solmaz TURKCAN, Forensic Psychiatry Unit, Bakırköy Psychiatric and Neurological Diseases Hospital, Istanbul Fatmahan ATALAR, Pediatric Endocrinology, Child Health Institute, Istanbul One of the recently identified risk gene in schizophrenia (SCH) is human ether-a-go-go related (herg) gene which belongs to a particular subtype known as H or Kv11 subfamily of the voltagegated potassium channels. Thyroid hormone, one of the major regulator of the Kv11.1 channels encoded by herg1 works through a signal transduction cascade involving the phosphatidylinositol 3-kinase (PI3K). Knowing the association between thyroid hormone dysfunction and schizophrenia, we aimed to investigate the effect of two polymorphisms; K897T (rs ) herg1 polymorphism, known to create a phosphorylation site leading to the channel activity inhibition, and Thr92Ala polymorphism (rs225014) in DIO2 gene, which converts the T4 to T3 an essential step in thyroid metabolism, either isolated or in combination in SCH. Design and Methods: The herg1 K897T and DIO2 Thr92Ala polymorphisms were genotyped by Taqman SNP method and by polymerase chain reaction restriction fragment length polymorphism analysis and they were also haplotyped in 78 SCH patients and 45 healthy controls. Results: rs SNP was associated with schizophrenia. SCH patients were more than four times as likely to carry the CT or TT genotypes compared to CC genotypes, indicating a dominant mode of inheritance. rs SNP showed no association with SCH. Analysis of rs225014/ rs haplotypes revealed a nearly significant association with SCH with A-T haplotype being almost 1.6 times more common in SCH patients. Conclusion: Our study provides further support for the importance of DIO2 and herg in schizophrenia. We identified a previously undescribed two-marker haplotype (A-T, P= 0.082) overrepresented in SCH patients compared to controls. It is possible that the rs SNP along with rs , result in a common haplotype that might contribute to schizophrenia susceptibility. As our participant numbers are limited for this preliminary study, we aim to replicate these results in larger cohorts to get adequate statistical power. Keywords: Schizophrenia, herg DIO2 polymorphism, haplotype 135

136 PP-45 ABBERANT EXPRESSION OF THE OBESITY HORMONE LEPTIN AND ITS RECEPTOR IN EPICARDIAL ADIPOSE TISSUE OF OBESE AND CAD PATIENTS Gokce AKAN, Medical Biology and Genetics, The Institute of Health Science, Istanbul Selcuk GORMEZ, Department of Cardiology, Acibadem Kadikoy Hospital, Istanbul Baris CAYNAK, Department of Cardiovascular Surgery, Istanbul Bilim University, Istanbul Zeliha YAZICI, Department of Pharmacology, Istanbul Cerrahpasa Medical School, Istanbul Demet GUNAY, Biochemistry Laboratory, Florence Nightingale Hospital, Istanbul Cihan DURAN, Department of Radiology, Istanbul Bilim University, Istanbul Belhhan AKPINAR, Department of Cardiovascular Surgery, Istanbul Bilim University, Istanbul Ugur OZBEK, Department of Genetics, Institute for Experimental Medicine, Istanbul Ahmet Sevim BUYUKDEVRIM, Department of Internal Medicine, Istanbul School of Medicine, Istanbul Fatmahan ATALAR, Department Growth-Development and Pediatric Endocrinology, Child Health Institute, Istanbul Backround and Aim: Leptin(LEP) and its receptor(lepr) play the central role in obesity pathogenesis. Leptin is almost exclusively produced mainly white adipose tissue (AT) and plays an important role in the regulation of energy balance.in this study we first analysed LEP c G>A and LEPR p.q223r polymorphisms and investigated their impact on leptin and adiponectin serum levels and the mrna expression levels of LEP and LEPR in epicardial AT (EAT), pericardial AT (PAT) and subcutaneous AT(SAT) in obese patients with CAD and obese patients with non-cad (controls).method:37 obese patients with CAD and 19 controls were included in this study.the peripheral blood, adipose tissues and serum were taken from the study group. LEP c.-2548g>a and LEPR p.q223r polymorphisms were studied by PCR-RFLP, leptin and adiponectin levels were measured by ELISA and the LEP and LEPR mrna expressions in EAT, PAT and SAT were studied by QRT-PCR. Result:The characteristics of the study group are summarized in Table1.As expected, the serum levels of leptin and adiponectin were statistically significant in obese patients with CAD compared to controls (p<0.05,respectively).there were no significant differences in allele frequencies or genotypes in both SNPs between the two groups.the LEP gene expression was found to be significantly increased in EAT, PAT, SAT of obese patients with CAD compared to controls.the LEPR gene expression level was found to be significantly increased only in EAT of obese patients with CAD compared to controls (p=0.002),although not significant, its expression level was found to be increased (three fold and two fold increase, respectively) in PAT and SAT.Conclusion:LEP and LEPR mrna levels were found to be significantly different in EAT of obese patients with CAD compare to controls.therefore leptin and leptin receptor need to be further studied in obesity associated with CAD especially in EAT, to be able to evaluate their prognostic values. Keywords: Obesity, CAD, LEP, LEPR, Adipose tissue 136

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138 PP-46 IL-32 IZOFORMS IN BEHCET DISEASE Nilgun SALLAKCI, Akdeniz University School of Medicine Pediatric Immunology-Allergy Department, Antalya Yahya KILINC, Akdeniz University School of Medicine Pediatric Immunology-Allergy Department, Antalya Ayse AKMAN-KARAKAS, Akdeniz University School of Medicine Department of Dermatology, Antalya Olcay YEGIN, Akdeniz University School of Medicine Pediatric Immunology-Allergy Department, Antalya Behcet disease (BD) is autoinflamatory disease, characterized by recurrent oral and genital ulcers, relapsing uveitis, mucocutaneus lesions, articular, neurologic, urogenital, intestinal manifestations. IL-32 is a proinflamatory cytokine that has six isoforms. In addition to it s proinflamatory effects, IL- 32 induce secretion of inflamatory cytokines such as IL-8, TNF-alpha which are known to play important role in Behcet disease. In this study, we investigated serum and supernatant level of IL- 32α, and mrna expression of various isoforms of IL-32 by reverse-transcriptase PCR, after human recombinant IL-17 (ril-17), ConcavalinA (ConA) and E.coli extract stimulation in 5 Behcet patients and 5 healthy controls. It was found that in healthy controls ril-17 did not induce any IL- 32 isoforms expression however in BD patients all isoforms were induced. In addition both healthy controls and BD patients expressed b isoform stronger than α and γ isoforms. We could not find any expression of θ isoform in none of the subjects. All Sera and supernatant IL-32 α level were found to be below the detection level. Our preliminary results show that Il-32 response to IL-17 stimulation is different in BD in comparison to healthy controls. Our study on IL-32 isoforms in BD both in activation and remission periods are being continued Keywords: behcet disease, IL-17 stimulation, Il-32 response 138

139 IL-32 isoforms GADPH 139

140 PP-47 ANTIBODY MEDIATED REJECTION IN RENAL TRANSPLANT RECIPIENTS Yasar CALISKAN, Division of Nephrology, Department of Internal Medicine, Istanbul Faculty of Medicine, Istanbul University, Istanbul Sebahat USTA, Department of Medical Biology, Istanbul Faculty of Medicine, Istanbul University, Istanbul Sonay TEMURHAN, Department of Medical Biology, Istanbul Faculty of Medicine, Istanbul University, Istanbul Yasemin OZLUK, Department of Pathology, Istanbul Faculty of Medicine, Istanbul University, Istanbul Cigdem KEKIK, Department of Medical Biology, Istanbul Faculty of Medicine, Istanbul University, Istanbul Halil YAZICI, Division of Nephrology, Department of Internal Medicine, Istanbul Faculty of Medicine, Istanbul University, Istanbul Isin KILICASLAN, Department of Pathology, Istanbul Faculty of Medicine, Istanbul University, Istanbul Fatma OGUZ SAVRAN, Department of Medical Biology, Istanbul Faculty of Medicine, Istanbul University, Istanbul Aydin TURKMEN, Division of Nephrology, Department of Internal Medicine, Istanbul Faculty of Medicine, Istanbul University, Istanbul Antibody Mediated Rejection in Renal Transplant Recipients Background: The aim of this study was to investigate the characteristics of antibody-mediated rejection (AMR), and significance of HLA-donor specific antibodies (DSA) in adult renal transplantation (RTx) patients. Methods: A total of 820 adult RTx cases were evaluated and 29 RTx recipients diagnosed as AMR by biopsy showed progressive deterioration in graft function and AMR as diagnosed by biopsy were enrolled. Results: Acute AMR was diagnosed by biopsy in 5 (%0.6) (mean age: 35 9 years, 3 male) patients on the median 8 day after living donor RTx. The median HLA mismatch number was 3. After the treatment for acute AMR, only 1 (20%) patient lost allograft during the follow up. Chronic active AMR (camr) was diagnosed by biopsy in 24 (%2.9) (mean age: years, 13 male, living/cadeveric: 19/5) patients on the mean months after RTx. The median HLA mismatch number was 3. The mean serum creatinine and proteinuria levels of patients at the time of diagnosis were mg/dl and g/day, respectively. Deposition of C4d in peritubular capillaries was found in 19 (79.1%) (diffuse (n=17, 70.8%), focal (n=2, 8.3%)) of the renal allograft biopsies. HLA antibodies were detected in 71% of the patients (17/24) taken at the time of camr, and 8 (33.3%) patients had class I and 14 (58.3%) patients had class II HLA antibodies. 82% (14/17) of these were HLA-DSA. Overall, 58.3% of the (14/24) of the patients had HLA-DSA. After the treatment given for camr, deterioration of renal allografts\' function was seen in 10 (41.6%). The antibody findings and C4d deposition were not correlated to serum creatinine, proteinuria and clinical outcome. Conclusion: Detection of HLA antibodies and deposition of C4d in peritubular capillaries are principal findings in camr after RTx, however they may not predict any deterioration of allograft function. Keywords: anti-hla, antibody, transplantation, rejection, humoral immunity, kidney 140

141 PP-48 CONSTRUCTION OF BISPECIFIC ANTIBODY WITH GOLD BINDING ABILITY FOR BIOSENSOR APPLICATIONS B. Koray BALCIOGLU, Immunogenetics, TUBITAK MRC, Gebze/Turkey Aylin OZDEMIR-BAHADIR, Immunogenetics, TUBITAK MRC, Gebze/Turkey H. Burak CALISKAN, Molecular Biology and Genetics, Istanbul Technical University, Istanbul/Turkey Duygu HINC, Immunogenetics, TUBITAK MRC, Gebze/Turkey Candan TAMERLER, Materials Science and Engineering, University of Washington, Seattle/USA Berrin ERDAG, Immunogenetics, TUBITAK MRC, Gebze/Turkey Purpose and aim: Hepatitis B virus (HBV) is one of the major causes of Chronic Hepatitis cirrhosis and liver cancer. Therefore diagnosis HBV infection is of great importance. Diagnosis of HBV by ELISA kit is a commonly used efficient method but it may take few hours to get results, for this reason the development of faster diagosis methods are necessary. Surface Plasmon Resonance (SPR) based biosensors are getting more and more promising for diagnosis purposes. In this study a new method for attaching antibodies onto gold-coated SPR chips is described. It consists on the development of bispecific recombinant antibodies with gold binding ability for biosensor applications. Methods: A recombinant antibody against HBsAg (Hepatitis B virus surface Antigen) previously developped in our Laboratory was cloned into the pqe2 expression vector. Gold binding peptide coding DNA sequence was then inserted into the vector. The fusion antibody was expressed in E.coli, purified and controlled by SDS PAGE and western blot analysis. The binding of the fusion antibody to gold, then the binding of HBsAg to the recombinant antibodies adsorbed on gold coated-chip were visualized by SPR. Results and discussion: Gold binding peptide DNA sequence was inserted at the 3 end of the anti-hbsag recombinant antibody. The cloning experiments were confirmed by DNA sequence analysis, SDS PAGE and western blot. The size of the construct was determined as 36kDa. SPR results showed that the bispecific recombinant antibody was binding to gold-coated chip and that HBsAg was binding to the bispecific antibody. In conclusion, the method described herein demonstrates that genetically engineered bispecific gold binding recombinant antibodies can be useful for SPR chip coating by a simple adsorption procedure. Keywords: Recombinant Antibody, Gold Binding Peptide Molecular Cloning, Fusion protein, Biosensor 141

143 PP-50 MICROSATELLITE POLYMORPHISM OF EQUINE MHC CLASS II GENE REGION Mehmet NIZAMLIOGLU, Biochemistry, Selcuk University Faculty of Veterinary Medicine, Konya Zafer BULUT, Biochemistry, Selcuk University Faculty of Veterinary Medicine, Konya Ercan KURAR, Genetics, Selcuk University Faculty of Veterinary Medicine, Konya Oya BULUT, Virology, Selcuk University Faculty of Veterinary Medicine, Konya Muge DOGAN, Biochemistry, Selcuk University Faculty of Veterinary Medicine, Konya Esma Gamze ILGAR, Biochemistry, Selcuk University Faculty of Veterinary Medicine, Konya Major Histocompatibility Complex (MHC) gene polymorphism in human and animals are known one the most polymorphic regions of the genome and have important functions in immune response and biology of immune system. MHC Class II region, located on chromosome 20 (ECA20), is highly polymorphic in horses. It was reported that there were 24 and 13 different alleles in the ELA-DQA and ELA-DQB loci respectively.. Due to their informativeness and high level of polymorphism, microsatellite markers are widely used in population genetic studies. In this study, five microsatellites located on ECA20 (Group-A: UMNe274, AHT018, TKY547, UMNe494, UMNe065) and 5 microsatellites on the other autosomes (Group-B: LEX33, HMS06, HMS02, HTG10, AHT04) were used and these groups were compared for marker polymorphism. Allel numbers (Na), observed (HO) and expected (HE) heterozygosities, and genetic relationship at each microsatellite locus were computed using package programs such as Genetix, GenAlEx6 and Structure. Numbers of alleles were observed as 7-17 in Group-A and 9-13 in Group-B markers. Ho values in Group-A and Group-B were observed as and , respectively. It was determined that He values were (Group-A) and (Group-B). As conclusion, high level of polymorphism was observed in microsatellite markers proximity to highly polymorphic MHC gene region. Informative characteristics of microsatellite markers can be used in studies of MHC region. Keywords: MHC, Microsatellite, Polymorphism, Horse 143

144 PP-51 EFFECT OF CD3 HAPLOINSUFFICIENCY ON PHENOTYPE, DEVELOPMENT AND FUNCTION OF TAB LYMPHOCYTES Marina MAZARIEGOS-LEÓN, Inmunologia, Universidad Complutense, Madrid Miguel MUÑOZ-RUIZ, Inmunologia, Universidad Complutense, Madrid Beatriz GARCILLÁN, Inmunologia, Universidad Complutense, Madrid Mario DELGADO, Biologia celular e inmunologia, Instituto Lopez Neyra, Granada Edgar FERNÁNDEZ-MALAVÉ, Inmunologia, Facultad de Medicina, Universidad Complutense, Madrid José R. REGUEIRO, Inmunologia, Facultad de Medicina, Universidad Complutense, Madrid OBJETIVE: Until the moment heterozygote mice for CD3g, CD3d and CD3e mutations have been considered as normal controls. Our findings suggest that they have a slight phenotype. In order to evaluate specific functions of CD3 chains, we analyze the effect of haploinsufficiency of CD3G (g+/-), CD3D (d+/-) or CD3E (e+/-) on development, phenotype and function of Tab lymphocytes. METHODS: Comparative flow cytometry, ELISA, Western-blot, functional assays and cecal ligation and punction (CLP). RESULTS: In periphery, it was shown (with gradient d g e) a gradual reduction of CD4+ population and an increase of the CD8+ one in haploinsufficient mice. It was also detected a solid reduction of TCRab expression, measured with anti-cd3 antibodies, on g+/- and e+/- T cells (70% of control with some antibodies and 50% with others), but not on d+/- T ones. The decrease of TCR expression correlated with decreased late functional parameters (anti- CD3-induced proliferation, IL-2 secretion and response to polymicrobial infection generated by CLP), but not with early ones (CD69 and CD25 induction) on g+/-, d+/- or e+/- T cells. Finally, defects on Akt activation in g+/- cells but not in d+/- were detected. CONCLUSION: CD3 chains have some different functions in TCR/CD3 complex expression and late functions, as well as in T cells development, since in haploinsufficient mice 1) TCRab expression and T cells development are more dependent on CD3g and e than on CD3d; 2) CD3g but not CD3d takes part in Akt activation; and 3) CD3g and e, and less CD3d, regulate IL-2 induction. Therefore, the hierarchy CD3d g e can be established, regarding its relative importance in haploinsufficient mice. 144

145 PP-52 PREVALENCE OF HLA-DRB1 ALLELES IN IRANIAN PATIENTS WITH RHEUMATOID ARTHRITIS Yadollah SHAKIBA, Immunology, TUMS, Tehran Susan KOOPAH, Immunology, TUMS, Tehran Ahmad Reza JAMSHIDI, Rheumatology, Shariati Hospital, Tehran Amir KIANI, Pharmacology/Toxicology, Molecular Diagnostic Research Center, Kermanshah Ahmad MASOUD, Immunology, TUMS, Tehran Ali Akbar AMIRZARGAR, Immunology, TUMS, Tehran Mohammad Hossein NICKNAM, Immunology, TUMS, Tehran Behrouz NIKBIN, Immunology, TUMS, Tehran BACKGROUND: Rheumatoid Arthritis (RA) is a chronic inflammatory disease with unknown etiology that affects about 1% of population. Association between different alleles of HLA-DRB1 and RA has been reported in many populations and geographical areas. The aim of the present study was to investigate the frequency of predisposing and protective HLA-DRB1 alleles in Iranian RA patients, comparing to normal population. METHODS: DNA was extracted from blood samples of 200 RA patients and 200 sex and age matched normal controls by standard phenol-chloroform method. The concentration and purity of DNA samples was determined by a spectrophotometer. HLA-DRB1 allele types were identified by polymerase chain reaction with sequence-specific primer (PCR-SSP). The frequency of HLA-DRB1 alleles was determined and analyzed by Epi Info- 7 software using chi-square test. RESULTS: The frequency of HLA-DRB1*04 (OR=1.59, P=0.018) and DRB1*10 (OR= 3.54, P=0.001) alleles was significantly higher in patients with RA, while the frequency of DRB1*07 (OR=0.54, P=0.025), DRB1*13 (OR=0.43, P=0.0002) and DRB1*16 (OR=0.42, P=0.02) alleles in these subjects was significantly lower than that in the control group. The frequencies of DRB1*01, DRB1*03, DRB1*08, DRB1*09, DRB1*11, DRB1*12, DRB1*14 and DRB1*15 were not significantly different between RA subjects and the control group. CONCLUSION: The data suggest that the DRB1*04 and DRB1*10 alleles are risk factors and DRB1*07, DRB1*13 and DRB1*16 are protective for RA in Iranian population. These data also suggests that the shared epitope may be involved in pathogenesis of RA in Iranian population. Keywords: HLA DRB1, Rheumatoid Arthritis, Iranian Population 145

146 PP-53 ASSOCIATION OF HLA-DRB1*14 AND DRB1*16 WITH MUSK-MYASTHENIA GRAVIS IN TURKISH PATIENTS Mahdi ALAHGHOLI HAJIBEHZAD, Immunology, DETAE, Istanbul Vuslat YILMAZ, Physiology, Istanbul Medical Faculty, Istanbul Yeşim GÜLŞEN PARMAN, Neurology, Istanbul Medical Faculty, Istanbul Fikret AYSAL, Neurology, Bakirkoy Training and Research Hospital for Mental Health and Neurological Diseases, Istanbul Piraye OFLAZER, Neurology, Istanbul Medical Faculty, Istanbul Feza DEYMEER, Neurology, Istanbul Medical Faculty, Istanbul Güher SARUHAN DIRESKENELI, Physiology, Istanbul Medical Faculty, Istanbul Aim: A strong association of relatively rare group of myasthenia gravis (MG), muscle-specific kinase antibody-positive (MP) has been reported with HLA-DR14-DQ5 followed by another report suggesting a role for DQ5. In this study, the probable association between HLA-DRB1*03,*14,*16 and MG with anti-acetylcholine receptor (AChR) antibody (AP), MP and both negative (SN) patient groups was to investigated. Methods: The study group includes 216 generalized MG patients (74 men/142 women, mean age: 42 years). Among the MG group, 119 patients had antibodies against AChR (AP), 44 were MP and 53 were negative for both antibodies (SN). About 13% of the patients had thymoma and 33.3% had a generalized disease with late onset (>40). The data of 250 healthy donors (HC, 109 men/141 women, mean age 29.8) was evaluated as control. Polymerase chain reaction with sequence specific primers (PCR-SSP) was used for typing of HLA-DRB1*03, DRB1*14, and DRB1*16 allele groups. Results: We found a highly significant association of both DRB1*16 and DRB1*14 with MP compared to HC (38.6 vs. 10.8%, p= 1.9x10-5, OR %CI and 32 vs. 11.6%, p , OR 3.55, 95%CI , respectively). DRB1*16 group was also more frequent in SN group (24.5%, p= 0.01, OR %CI ). On contrary, HLA-DRB1*03 was found to be negatively associated with MP patients (2.3 vs. 18.4%, p=0.006, OR %CI ). In this selected group of MG patients with a dominance of rare subgroups, the combination of disease related HLA-DRB1*16 and DRB1*14 was even more strongly associated with the disease in MP (63.6 vs. 22%, p= 9.63x10-8 OR %CI ). Conculsion: This study confirm the highly significant association of HLA-DRB1*16 and HLA-DRB1*14 alleles, especially in MP MG and provides a replication of these results in Turkish population. 146

147 PP-54 EVALUATION OF MULTIPLE SCLEROSIS GENETIC COMPLEXITY BASED ON THE ANALYSIS OF THREE IMMUNE-ASSOCIATED POLYMORPHIC SYSTEMS Snezhina MIHAILOVA, Department of Clinical Immunology, University Hospital "Alexandrovska", Sofia Milena IVANOVA, Department of Clinical Immunology, University Hospital "Alexandrovska", Sofia Anastassia MIHAYLOVA, Department of Clinical Immunology, University Hospital "Alexandrovska", Sofia Derek MIDDLETON, Transplant Immunology, Royal Liverpool University, Liverpool Ellisaveta NAUMOVA, Department of Clinical Immunology, University Hospital "Alexandrovska", Sofia Aim: Multiple sclerosis (MS) is a complex genetic disease with autoimmune component. The aim was to evaluate the role of three immune-related genetic systems in MS etiology cytokine genes, NK genes and their HLA ligands based on the polymorphisms investigation. Methods: 104 healthy Bulgarians and 58 relapsing-remitting MS patients were assayed for HLA-B, C, DRB1, DQB1, 16 KIR genes, TNF-a, TGF-b, IL-10, IL-6 and IFN-g by PCR-SSP and PCR-SSOP methods. Results: The strong MS predisposing effect of DRB1*1501- DQB1*0601 was confirmed in our population but additionally DQB1*02 allele and DRB1*13- DQB1*03 haplotype also showed a positive association with the disease. Statistically significant increased frequencies of B*15 (p=0.036), B*73 (p=0.007) and HLA-Cw*15 (p=0.016) were found in MS patients. Individuals homozygous for group 2 HLA-C ligands were more frequent in the patient s group (p=0.05). In addition a tendency for prevalence of 2DS1 gene in the MS in comparison to the healthy subjects (46.3% vs. 33.3%) and an increased frequency of KIR2DS1 in combination with their group 2 HLA-C ligands (p=0.039, OR=3.52) were observed. The investigation of cytokine gene polymorphism revealed statistically significant increase in the CC genotype of IL and -592 SNPs coupled with a decreased frequency of the TGF-b +915 CG genotype in MS patients (Pc<0.05). Conclusion: Data implies that some HLA alleles are predisposing for MS in the Bulgarian population. Furthermore, distinct KIR or KIR/HLA-ligand combinations may be relevant to the development of autoimmune disease in the manner that the activation overrides inhibition of NK and NKT cells. Additionally polymorphic variations of two of the major anti-inflammatory cytokines, IL-10 and TGF-b, may play a role in MS susceptibility determining dysregulation in Th2 profile. The cumulative effect of studied genetic markers seems to be important event for disease etiology but which of them play primary role remains controversial. 147

149 PP-56 HLA-DRB1 ALLELES AND PRODUCTION OF ANTI-CCP ANTIBODY IN IRANIAN PATIENTS WITH RHEUMATOID ARTHRITIS Yadollah SHAKIBA, Immunology, TUMS, Tehran Delnia ARSHADI, Immunology, TUMS, Tehran Ahmad Reza JAMSHIDI, Rheumatology, Shariati Hospital, Tehran Amir KIANI, Pharmacology/Toxicology, Molecular Diagnostic Research Center, Kermanshah Ahmad MASOUD, Immunology, TUMS, Tehran Ali Akbar AMIRZARGAR, Immunology, TUMS, Tehran Mohammad Hossein NICKNAM, Immunology, TUMS, Tehran Behrouz NIKBIN, Immunology, TUMS, Tehran Background: The aim of the present study was to evaluate the associations between HLA-DRB1 alleles with production of anti cyclic citrullinated peptide (anti-ccp) antibody in Iranian patients with rheumatoid arthritis (RA). Methods: DNA We extracted from blood samples of 200 RA patients and 220 sex and age matched controls. DRB1*alleles were determined by polymerase chain reaction sequence-specific primer (PCR-SSP) method. Serum levels of anti-ccp antibody were measured by commercial kits using an ELISA method. Data analyzed by Epi Info-7 software. Results: The results showed that HLA-DRB1*04 and *10 alleles were associated with increased risk of RA in Iranian patients (P=0.018 and P=0.001 respectively). We also showed that both of these alleles were associated with anti-ccp antibody production in patients (p=0.045 and p=0.03 respectively). Discussion: This study indicates that RA predisposing alleles in Iran are also associated with anti-ccp antibody production in these patients. It seems that HLA-DRB1*04 and *10 alleles involve in pathogenesis of RA in Iranian population by combination of different mechanisms. Keywords: Rheumatoid arthritis, Iranian population, Anti-CCP, HLA-DRB1 149

150 PP-57 TIR-DOMAIN-CONTAINING ADAPTOR PROTEIN GENE TIRAP S180L POLYMORPHISM IN BEHCET S DISEASE Gorkem TURUNC, Physiology, Istanbul Medical Faculty, Istanbul Doga COSKUN, Physiology, Istanbul Medical Faculty, Istanbul Fatma ALIBAZ-ONER, Rheumatology, Marmara University Medical School, Istanbul Sibel P. YENTUR, Physiology, Istanbul Medical Faculty, İstanbul Tulin ERGUN, Dermatology, Marmara University Medical School, Istanbul Haner DIRESKENELI, Rheumatology, Marmara University Medical School, Istanbul Guher SARUHAN-DIRESKENELI, Physiology, Istanbul Medical Faculty, Istanbul Objective: Genetic variations in innate immune system may have a role in the susceptibility to Behcet s disease (BD). Toll-like receptors (TLR) are the major pattern-recognition receptors for anti-bacterial innate immunity. MyD88-adapter-like molecule (TIRAP, toll-interleukin-1 receptor domain containing adaptor protein, MAL) have a regulatory role for TLR2/4 signalling. A single nucleotide polymorphism (SNP) of TIRAP causing an amino acid change (Serine 180 leucine, S180L) is shown to have an anti-inflammatory effect in infections such as tuberculosis. In this study, TIRAP S180L is investigated in BD. Methods. The association of TIRAP SNP (rs , C/T, S180L) is investigated on 196 patients with BD (92 women/104 men, mean age: 36,3 years) and 161 healthy controls (66 women/95 men, mean age: 32 years) by using PCR-SSP. The patients and controls were screened for the presence of HLA-B*51 by PCR-SSP as well. Results: In BD group the distribution of genotypes was not significantly different from the controls: CC genotype was detected in 74 vs. 81.4%, CT genotype in 22.4 vs. 17.4% and TT genotype in 3.6 vs. 1.2% of patients and controls, respectively. However the reported increase of T allele frequency was observed as a trend also in this patient population: 0.19 vs (p= 0.05, OR %CI ). A high presence of HLA-B*51 was found in the patient population (OR=5.68, 95% CI: ). When analyzed according to HLA-B*51 presence, no significant association of the TIRAP S180L polymorphism was present either in the HLA-B*51 positive or in HLA-B*51 negative groups. Conclusion: TIRAP (rs ) gene polymorphism, which was previously shown to be associated with BD in a Caucasian population, has been not replicated in this population from Turkey, although a trend was observed. A broader screening of genetic polymorphisms in other molecules of innate immunity is warranted. This study is supported by Istanbul University Research Fund (BAP). Keywords: TIRAP, S180L polymorphism, Behcet's Disease 150

151 PP-58 CLONING AND PROKARYOTIC EXPRESSION OF HEPATITIS C VIRUS E1 ENVELOPE GLYCOPROTEIN Özge ÖZTUNA, Microbiology, Health Sciencies, Izmir İ.Mehmet Ali ÖKTEM, Microbiology, Medicine Faculty, Izmir Y.Hakan ABACIOĞLU, Microbiology, Medicine Faculty, Izmir AIM: Hepatitis C virus (HCV) infection is a major public health issue. HCV has a propensity to develop chronic infections which may proceed to cirrhosis and hepatocellular carcinoma. One of the hallmarks of the chronic infection is the quasispecies nature of the viral population which favors the selection of variants that may escape the host immune responses. Envelope glycoproteins (E1 and E2) are constantly under selective pressures resulting in the selection of viral variants that can escape antibody responses. E1 protein also plays important roles in viral attachment and entry into target cells as well as in the modulation of host immune responses. In this study, we report the cloning and prokaryotic expression of a genotype 1b HCV E1 molecule. METHODS: For the expression of HCV E1 protein in E. coli, full-length and C-terminally truncated E1 sequences from a clinical isolate of subtype 1b virus were amplified by PCR. Resulting fragments were cloned into pqe30 vector and subsequently expressed in E. coli as His-tagged proteins. The E1 proteins were characterized by SDS-PAGE. The immunologic reactivity of the proteins were evaluated by western blot (WB) analysis using anti-hcv positive serum samples. RESULTS: Full-length and C- terminally truncated E1 were successfully expressed in prokaryotic system as hexa-histidine-tagged recombinant fusion proteins. The proteins self-assembled into dimers and trimers as shown by PAGE and WB PAGE analyses. Recombinant E1 proteins were also immunologically reactive using polyclonal sera from HCV infected patients. CONCLUSION: Prokaryotic expression of full-length viral membrane proteins can be difficult. We report here the successful expression of the immunologically reactive full-length and truncated E1 proteins for the first time in Turkey. The re1 proteins can be important tools fur further diagnostic and immunological studies. Keywords: HCV envelope protein 1, Recombinant proteins, Escherichia coli 151

152 PP-59 IL23R GENE POLYMORPHISM IS ASSOCIATED WITH BEHCET S DISEASE IN A SECOND COHORT IN TURKEY Ayça DUMAN, Physiology, Istanbul Medical Faculty, Istanbul Vusale ABBASOVA, Immunology, Istanbul Medical Faculty, Istanbul Fatma ALIBAZ-ONER, Rheumatology, Marmara Medical School, Istanbul Vuslat YILMAZ, Physiology, Istanbul Medical Faculty, Istanbul Tulin ERGUN, Dermatology, Marmara Medical School, Istanbul Haner DIRESKENELI, Rheumatology, Marmara Medical School, Istanbul Guher SARUHAN-DIRESKENELI, Physiology, Istanbul Medical Faculty, Istanbul Objective: Both genetic and environmental factors are implicated in Behcet s disease (BD) pathogenesis. Recently two genome-wide association studies identified IL23R-IL12RB2 region as a BD susceptibility marker in Turkey and Japan. Another study from China described a strong association of a SNP of IL23R, rs , with BD as well. In this study, we investigated this intronic polymorphism of IL-23R gene (IL23R), in a second cohort from Turkey. Methods: The study was designed as a case-control study with 217 BD patients (Male/Female: 115/102, mean age: 35.8 years) and 242 healthy controls (M/F: 130/112, mean age: 32.0 years). The DNA samples from patients and the controls were genotyped by PCR-RFLP method for IL23R (G/A, rs ) gene polymorphism. Polymorphic region was amplified by PCR and digested with BsuR I enzyme for G allele. The patients and controls were typed for the presence of HLA-B*51 by PCR-SSP. Results. In BD group the distribution of genotypes was significantly different than in controls: GG genotype and G allele were increased in BD (58.5 vs. 40.9%, p= , OR=2.03, 95% CI: and 333/434 vs. 320/484, p= , OR=1.69, 95% CI: , respectively). A strong presence of HLA-B*51 was observed in the patient population (OR=6.26, 95% CI: ). When compared according to HLA-B*51, the association of the GG genotype of IL23R SNP was evident in the HLA-B*51 positive patients as well, but was stronger in HLA- B*51 negative patients (OR=1.97, 95% CI: and OR=2.4, 95% CI: , respectively). Subgroup analysis according to ocular, mucocutaneous or vascular involvement did not reveal any significant differences. Conclusion: The association of IL23R (rs ) gene polymorphism with BD has been replicated in Turkey and this association was independent of HLA-B*51. The Study was supported by Istanbul University Research Fund (BAP). 152

153 PP-60 THE ASSOCIATION OF HLA-B51 EXPRESSION WITH ENDOPLASMIC RETICULUM STRESS Fulya COŞAN, Department of Immunology, Institute for Experimental Medicine (DETAE), Istanbul Zeliha EMRENCE, Department of Genetics, Institute for Experimental Medicine (DETAE), Istanbul Hülya AZAKLI, Department of Genetics, Institute for Experimental Medicine (DETAE), Istanbul Sema SIRMA, Department of Genetics, Institute for Experimental Medicine (DETAE), Istanbul Esin ÇETİN AKTAŞ, Department of Immunology, Institute for Experimental Medicine (DETAE), Istanbul Günnur DENİZ, Department of Immunology, Institute for Experimental Medicine (DETAE), Istanbul Duran ÜSTEK, Department of Genetics, Institute for Experimental Medicine, (DETAE), Istanbul Aim: Endoplasmic reticulum (ER) is an organel, where the proteins are folded. Misfolding of the proteins causes the endoplasmic reticulum stress. Unfolded protein response occurs for compensating of ER stress. It was shown the UPR molecules play a role in the pathogenesis of several diseases and it was suggested, that HLA-B*27, the major genetic susceptibility for ankylosing spondylitis, is associated with UPR. HLA-B*51, which has the same Bw4 region and associated with Behçet s disease (BD), may have similar misfolding pattern. In this study, the association between the expression of HLA-B*51 and UPR molecules were investigated. Method: HLA-B*51 expressing monocyte cell line THP-1 monocytes and from THP-1 derived macrophages were stimulated with LPS, ATP, IFN- and Tunicamycin. After 8 and 24 hrs stimulation, mrnas were isolated and the expression of ATF6, IRE1, PERK, XBP-1, BIP were performed with quantitative PCR. In addition, HLA-B*51 (+), (-) and B*27(+), (-) PBMCs from 8 individuals were cultured, with the same agents stimulated and quantitative PCR was performed. The correlation analysis was performed with using SPSS Results. There was a correlation between HLAexpression and IRE1 (r(12)=.957, p<0,01), PERK (r(12)=.974, p<0,01), ATF6 (r(12)=.952, p<0,01), BİP (r (12)=.610, p<0,05) in THP-1 monocytes. A strong correlation also found with BİP (r(12)=.808, p<0,01) and ATF6 (r(12)=.626, p<0,05) expression in THP-1 derived macrophages. In HLA-B51 (+) PBMC cell culture the correlation was found with PERK (r(12)=.535, p<0,05) and ATF6 (r(12)=.622, p<0,05) expression. There was no similarity in the expression of UPR molecules in HLA-B*51 (-) cell cultures. In HLA-B*51 expressing PBMC derived macrophages, a correlation was detected between HLA and IRE1 (r(12)=.746, p<0,01), PERK (r(12)=.696, p<0,01) and ATF6 (r(12)=.558, p<0,05). Increased HLA expression was also found correlated with PERK (r(12)=.903, p<0,01) and ATF6 (r(12)=.706, p<0,01) in HLA-B*27(+) PBMC culture. In HLA-B*51 and B*27 (-) cell cultures did not show any strong UPR response to different stimulating agents. Conclusion: Our data reveals that it is a strong association between the expression of HLA-B*51 and specific UPR molecules after stimulation, while the HLA-B*51 and B*27 negative cultures did not respond to stimulant agents in similar pattern. In the monocytes HLA-B*51 expression is found especially correlated with PERK and ATF6. This finding may show the role of ER stress in BD pathogenesis. 153

156 PP-62 PEPTIDE-BASED MONOCLONAL ANTIBODY PRODUCTION AGAINST β-actin PROTEIN Nazila AMINI, Monoclonal Antibody Research Center, Avicenna Research Institute, Tehran Mohadeseh NAGHI VISHTEH, Monoclonal Antibody Research Center, Avicenna Research Institute, Tehran Sayeh KHANJANI, Reproductive biotechnology Research Center, Avicenna Research Institute, Tehran Forough TORABI, Reproductive biotechnology Research Center, Avicenna Research Institute, Tehran Omid ZAREI, Department of Pharmaceutical Biotechnology,Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz Reza HADAVI, Monoclonal Antibody Research Center, Avicenna Research Institute, Tehran Hodjattallah RABBANI, Monoclonal Antibody Research Center, Avicenna Research Institute, Tehran Mahmood JEDDI-TEHRANI, Monoclonal Antibody Research Center, Avicenna Research Institute, Tehran Aim: Actin is one of the most widely studied structural and multifunctional proteins in eukaryotic cells that has important roles in many cell functions. The aim of this study was producing monoclonal antibodies against a synthetic peptide derived from N-terminal region of β-actin protein in order to use in different applications. Methods: A synthetic peptide derived from β-actin was designed and used to immunize two Balb/c mice. The mice were bled and serum ELISA assay was performed to evaluate the immunization efficiency. In order to produce hybridoma cells, spleen of the immunized mouse with higher serum antibody titer was removed at sterile conditions and spleenocytes were fused to mouse myeloma SP2/0 cell line followed by seeding into 96-well plates in the presence of selective HAT medium. Clone formation was examined daily and the presence of antibody against the immunizing peptide was determined by ELISA. The produced antibody was purified from supernatant of cultured selected-clones by affinity chromatography column prepared by coupling the immunizing peptide to CNBr-activated sepharose 4B. The quality of the purified antibody was evaluated by SDS-PAGE. Its specific interaction with the immunizing peptide was determined by ELISA. Immunoreactivity of the antibody was also tested by immunocytochemistry (ICC) as well as immunohistochemistry (IHC) and Western blotting (WB) in a panel of organisms with different origins. All experiments were repeated using a commercial antibody for comparison. Results: The results showed that the antibody had desired purity and reactivity with the peptide-bsaconjugate in SDS-PAGE and ELISA, respectively. It could also detect a band of about 45 kda corresponding to β-actin protein in WB. Moreover, the antibody could react with β actin in ICC and IHC. Conclusion: The anti-β-actin antibody may be used as an internal control for WB and may serve as a tool in different experiments such as ELISA, ICC and IHC. 156

160 PP-64 HEMOGLOBIN A2 (HBA2) NEGATIVELY CORRELATES WITH POST-PARTUM ATTACKS IN BIPOLAR DISORDER IN CONCERT WITH LESSENED INFLAMMATION AND HBF Meric A. ALTINOZ, Molecular Biology and Genetics, Halic University, Istanbul Bahri İNCE, Psychiatry, Urfa Training and Research Hospital, Urfa Gunnur DENIZ, Immunology, DETAE, Experimental Medicine and Research Institute, Istanbul Zeynep L. CIRAKLI, Biochemistry, Sadi Konuk Training and Research Hospital, Istanbul Latif ALPKAN, Psychiatry, Bakirkoy Training and Research Hospital, Istanbul Kursat ALTINBAS, Psychiatry, Bakirkoy Training and Research Hospital, Istanbul E. Timucin ORAL, Department of Psychology, Istanbul Commerce University, Istanbul Bipolar disorder is a psychiatric disorder with manic and depressive attacks. Many attempts have been made to understand the opposite patterns of disease, yet none was proven as succesful. Here we refer to hypoxia/inflammation/minor hemoglobin (Hb A2) to fetal hemoglobin (HbF) ratio as a likely explanation of bipolar disorder. HbA2 elevates after myocardial infarcts and lessens with recovery. HbA2 also elevates by climbing to higher altitudes and binds stronger to CO2 and CO2-binding erythrocyte Band-3 protein than major hemoglobin. Fossorial moles resist hypoxic hypercapnia by delta-globin s CO2 removal properties. In bipolar disorder, patients go through episodic events of cerebral hypoxia, yet no study has questioned, whether HbA2 or HbF associate with disease phenotypes. HbF has dual characteristics in human pathology, its increase until a certain level is beneficial in sickle cell anemia via prevention of HbS polymers, yet higher levels of HbF impairs wound healing. Moreover, gamma-globin chain of HbF increases inflammation and Th1 type cytokines. In bipolar disorder, a dominance of Th1 type cytokines occurs during episodes. Here, via analysing the hemoglobin electrophoreses of 120 euthymic bipolar patients, we found that level of HbF is higher in bipolar patients with a positive family history of any psychotic disorder. In opposite, HbA2 negatively correlated both with the presence and number of post-partum attacks in bipolar women. Moreover, HbA2 was lower in patients progressing with pure manic attacks in comparison to patients with both manic and depressive episodes. Hemogram indices showed a tendency of lowered inflammation with HbA2 increase. We propose that elevated Th1 to Th2 ratio and proinflammatory stage of bipolar disease is the result of hypoxia episodes and decreased HbA2 to HbF ratios, which we will address in further studies. 160

161 PP-65 COTREATMENT WITH THE HISTONE DEACETYLASE INHIBITOR SUBEROYLANILIDE HYDROXAMIC ACID (SAHA) INCREASES METHOTREXATE-INDUCED APOPTOSIS OF K562-CHRONIC MYELOID LEUKEMIA Ergul MUTLU ALTUNDAG, Biochemistry, Marmara University Medical School, Istanbul Cenay CIHAN, Biochemistry, Marmara University Medical School, Istanbul Ahmet CINGOZ, Biochemistry, Marmara University Medical School, Istanbul Yavuz TAGA, Biochemistry, Marmara University Medical School, Istanbul Semra KOÇTÜRK, Biochemistry, Dokuz Eylul University, Izmir Histone deacetylase (HDACs) regulate histone acetylation by catalyzing the removal of acetyl groups on the NH2-terminal lysine residues of the core nucleosomal histones. Modulation of the acetylation status of core histones is involved in the regulation of the transcriptional activity of certain genes. HDAC activity is generally associated with transcriptional repression. Methotrexate is part of a general group of chemotherapy drugs known as anti-metabolites. It prevents cells from using folate to make DNA and RNA. Because cancer cells need these substances to make new cells, methotrexate helps to stop the growth of cancer cells. K562, a human chronic myelogenous leukaemia cell line that can differentiate into recognisable progenitors of the erythrocyte, granulocyte and monocytic series. The histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA) is being evaluated for methotrexate-induced chronic myelogenous leukemia (CML) and has multiple cellular effects, including the induction of apoptosis. HDACi arrests the cell cycle in rapidly proliferating tumor cells and promote their apoptosis. In this study we have examined the apoptotic effects of SAHA and methotrexate on K562 cell line. Cytotoxicity was determined by WST-1 assay. Apoptosis was measured by Annexin-PI and cell cycle assay. We demonstrated apoptotic cells by immunohistochemistry and immunofluorescence staining. The study clearly showed the dose dependent cytotoxic effect of methotrexate and SAHA in K562 cells. These findings indicate that cotreatment with SAHA enhances the cytotoxic effects of methotrexate-induced apoptosis of K562-chronic myeloid leukemia cell line and may have activity against methotrexate refractory chronic myeloid leukemia. Keywords: K562, SAHA, Methotrexate, Apoptosis 161

162 PP-66 FUNCTIONAL ELIMINATION OF DOUBLE-STRANDED DNA-SPECIFIC B LYMPHOCYTES SUPPRESSES DISEASE ACTIVITY IN SCID MODEL OF MOUSE LUPUS Stela CHAUSHEVA, Immunology, Institute of Microbiology, Bulgarian Academy of Sciences, Sofia Nikolina MIHAYLOVA, Immunology, Institute of Microbiology, Bulgarian Academy of Sciences, Sofia Vera GESHEVA, Immunology, Institute of Microbiology, Bulgarian Academy of Sciences, Sofia Nikola KEREKOV, Immunology, Institute of Microbiology, Bulgarian Academy of Sciences, Sofia Kalina NIKOLOVA, Immunology, Institute of Microbiology, Bulgarian Academy of Sciences, Sofia Andrey TCHORBANOV, Immunology, Institute of Microbiology, Bulgarian Academy of Sciences, Sofia Objectives: Self-specific B cells play a main role in pathogenesis of Systemic lupus erythematosus (SLE). The elimination of B and T cells involved in the pathological immune response is a reasonable approach for effective therapy of SLE. In the present study we established an autoimmune model by transferring purified B and T cells from MRL/lpr mice to SCID mice and tested the effects of the chimeric molecule that selectively targeted pathological autoreactive B- lymphocytes. Methods: The protein chimeric molecule was constructed by coupling an DNAmimotope peptide DWEYSVWLSN to a monoclonal anti-cd32 (FcγRIIb) antibody. This engineered molecule is able to cross-link cell surface immunoglobulin with the inhibitory FcγRIIb on DNA-specific B cells. Female SCID mice were transferred with isolated B+T cells from MRL/lpr mice and the animals were treated with DNA- peptide chimera. Cytokine assays, ELISpot, Signal transduction, Proliferation assay, ELISA and FACS analyses were also performed. Results: The specific elimination of the DNA-specific B cells in MRL-transferred SCID mice restricts not only anti-dsdna IgG production, but also autoreactive T cell activation and proliferation. In contrast, untreated MRL-transferred SCID mice experienced an increase of disease-associated antibody levels and developed glomerulonephritis similar to intact MRL/lpr mice. Conclusions: In the present study we report a possible way to restrict the communication between autoimmune B and T cells, leading to suppression of lupus syndrome in MRL/lpr cell-transferred SCID mice. The functional elimination of autoantigen-specific B cells leaves autoreactive T cells alone without potency of prolonged pathogenetic effects. In the present transferred SCID model of mouse lupus we have a possibility to study post-elimination disease processes and autoreactive T cell behavior after treatment with the protein-engineered antibody. Keywords: autimmunity, humanized murine model, SLE, chimeric antibody 162

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